B. subtilis Sec and Srp Systems Show Dynamic Adaptations to Different Conditions of Protein Secretion.

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

First Report of 'Candidatus Phytoplasma citri' related strain (16SrII-C subgroup) associated with phyllody disease of cumin in India.

Cumin (Cuminum cyminum L.), is an important export-oriented seed spice crop for India. Cumin is popularly used for flavouring food, including soups, pickles and vegetables, and for herbal medicine. India is the largest producer, consumer and exporter of cumin seed with an annual production of 0.795 million tones over an area of 1.09 million hectares. During 2020-21, India exported about 0.299 million tons of cumin worth of Rs 33280 million (Anonymous, 2021). Recently, phytoplasma suspected symptoms were observed in cumin at Agricultural Research Station, Mandor, Jodhpur, Rajasthan, India from 2019. The symptoms related to phytoplasma infection were first recorded after 70-75 days of sowing in the month of January of the years 2019 to 2022. The major symptoms recorded were yellowing, phyllody, witches-broom, yellowing and deformed elongated seeds. Disease incidence was recorded as 0.25-1.0%, 0.5-1.5%, 0.5-2.5 % and 0.5-10.6% during the years 2019, 2020, 2021 and 2022, respectively using quadrate method. In 2022, among different genotypes assessed GC 4, MCU 73, MCU 105, and MCU 32 exhibited lower disease incidences ranging from 0.5% to 1.5%, while MCU 78 recorded the highest disease incidence at 10.6%. To detect the association of phytoplasma with symptomatic cumin samples, genomic DNA was extracted from four representative cumin genotypes (CuPP-MND-01 to CuPP-MND-04) and one asymptomatic cumin plant using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) (Schneider et al., 1995; Gundersen and Lee, 1996) and secA gene specific primers (SecAfor1/SecArev3 followed by nested PCR primers SecAfor5/ SecArev2) (Hodgetts et al. 2008; Bekele et al. 2011). The amplicons of ∼1.25 kb with 16S rRNA gene and ∼600 bp with secA gene specific primers were amplified in all symptomatic cumin plant samples and positive control of brinjal little leaf. PCR amplified products from the four selected positive samples (CuPP-MND-01 to CuPP-MND-04) of 16S rRNA gene and secA gene, were sequenced from both ends. The 1,245 bp sequences were deposited in GenBank (OQ299007-10), which showed 100% sequence identity with each other and 99.4% identity with 'Candidatus Phytoplasma citri' reference strain (GenBank accession: U15442) (Rodrigues Jardim et al. 2023). The phylogenetic analysis and virtual RFLP analysis using 17 restriction enzymes of 16S rRNA gene sequences through iPhyclassifier allowed affiliating the cumin phytoplasma strains with 16SrII-C subgroup strain with a similarity coefficient of 1 to the reference pattern of 16Sr group II, subgroup C (GenBank accession: AJ293216) (Zhao et al. 2009). In addition, the phylogenetic analysis of the secA gene-based sequences (OQ305073-76) further confirmed the close association of 16SrII-C group phytoplasmas with phyllody and witches' broom disease of cumin. Earlier 16SrII-C subgroup phytoplasma has been reported from various crops and weeds in India (Rao et al. 2021). However, no phytoplasma association has been reported earlier with cumin in India and abroad. To the best of our knowledge, this is the first report on the association of 16SrII-C group phytoplasma causing phyllody, witches' broom in cumin genotypes. This report has economic and epidemiological implications and needs immediate attention to reduce export losses due to phytoplasma disease in cumin and to prevent the potential spread to other crops.

Dynamic coupling of fast channel gating with slow ATP-turnover underpins protein transport through the Sec translocon.

The Sec translocon is a highly conserved membrane assembly for polypeptide transport across, or into, lipid bilayers. In bacteria, secretion through the core channel complex-SecYEG in the inner membrane-is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the conformational state of SecYEG throughout the ATP hydrolysis cycle of SecA. We show that the SecYEG channel fluctuations between open and closed states are much faster (~20-fold during translocation) than ATP turnover, and that the nucleotide status of SecA modulates the rates of opening and closure. The SecY variant PrlA4, which exhibits faster transport but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore and thereby enhancing translocation efficiency. Thus, rapid SecYEG channel dynamics are allosterically coupled to SecA via modulation of the energy landscape, and play an integral part in protein transport. Loose coupling of ATP-turnover by SecA to the dynamic properties of SecYEG is compatible with a Brownian-rachet mechanism of translocation, rather than strict nucleotide-dependent interconversion between different static states of a power stroke.

Autologous serum eye drops diluted with 0.5% methylcellulose and 0.9% saline solution: a clinical comparative study / Colírio de soro autólogo diluído com metilcelulose 0,5% e solução salina 0,9%: um estudo clínico comparativo

ABSTRACT Purpose: This clinical study compared autologous serum eye drops diluted with 0.5% methylcellulose and 0.9% saline solution. The subjective criteria for symptom improvement and the objective clinical criteria for response to therapy were evaluated. Methods: This longitudinal prospective study enrolled 23 patients (42 eyes) with persistent epithelial defects or severe dry eye disease refractory to conventional therapy who had been using autologous serum 20% prepared with methylcellulose for > 6 months and started on autologous serum diluted in 0.9% saline solution. The control and intervention groups consisted of the same patients under alternate treatments. The subjective criteria for symptom relief were evaluated using the Salisbury Eye Evaluation Questionnaire. The objective clinical criteria were evaluated through a slit-lamp examination of the ocular surface, tear breakup time, corneal fluorescein staining, Schirmer's test, rose Bengal test, and tear meniscus height. These criteria were evaluated before the diluent was changed and after 30, 90, and 180 days. Results: In total, 42 eyes were analyzed before and after 6 months using autologous serum diluted with 0.9% saline. No significant differences were found in the subjective criteria, tear breakup time, tear meniscus, corneal fluorescein staining, or rose Bengal test. Schirmer's test scores significantly worsened at 30 and 90 days (p=0.008). No complications or adverse effects were observed. Conclusions: This study reinforces the use of autologous serum 20% as a successful treatment for severe dry eye disease resistant to conventional therapy. Autologous serum in 0.9% saline was not inferior to the methylcellulose formulation and is much more cost-effective.

RESUMO Objetivo: Este estudo comparou o colírio de soro au tólogo manipulado com metilcelulose a 0,5% com solução salina 0,9%. Critérios subjetivos de melhora dos sintomas e critérios clínicos objetivos para resposta à terapia foram avaliados. Métodos: Este estudo prospectivo longitudinal envolveu 23 pacientes (42 olhos) com defeitos epiteliais persistentes ou doença de olho seco grave refratária à terapia convencional que usavam colírio de soro autólogo 20% preparado com metilcelulose por mais de 6 meses e iniciaram soro autólogo diluído em solução salina 0,9%. Os grupos controle e intervenção consistiam dos mesmos pacientes sob tratamentos alternados. Os critérios subjetivos para o alívio dos sintomas foram avaliados usando o Salisbury Eye Evaluation Questionnaire. Os critérios objetivos foram avaliados por meio de exame em lâmpada de fenda incluindo: tempo de ruptura da lágrima, coloração da córnea com fluoresceína, teste de Schirmer, coloração com rosa bengala e altura do menisco lacrimal. Esses critérios foram avaliados antes da troca do diluente e após 30, 90 e 180 dias. Resultados: Um total de 42 olhos foram analisados antes e após 6 meses usando soro autólogo diluído com solução salina 0,9%. Nenhuma diferença significativa foi encontrada nos critérios subjetivos, tempo de ruptura da lágrima, menisco lacrimal, coloração com fluoresceína ou rosa bengala. Os resultados dos testes de Schirmer pioraram significativamente em 30 e 90 dias (p=0,008). Não foram observadas complicações ou efeitos adversos. Conclusões: Este estudo reforça o uso do colírio de soro autólogo 20% como um tratamento de sucesso para a doença do olho seco grave resistente à terapia convencional. O soro autólogo diluído em solução salina a 0,9% não foi inferior à formulação de metilcelulose.

Catheter-related bloodstream infection caused by Tsukamurella tyrosinosolvens identified by secA1sequencing in an immunocompromised child: a case report.

BACKGROUND: Tsukamurella spp. are obligate aerobic, gram-positive, non-motile, and slightly acid-fast bacilli belonging to the Actinomycetes family. They share many characteristics with Nocardia, Rhodococcus, Gordonia, and the rapidly growing Mycobacterium species. Therefore, standard testing may misidentify Tsukamurella spp. as another species. Accurate and rapid diagnosis is critical for proper infection management, but identification of this bacterium is difficult in the standard laboratory setting. CASE PRESENTATION: A bloodstream infection caused by a gram-positive bacterium and related to a central venous catheter was identified in an immunocompromised 2-year-old girl. Tsukamurella tyrosinosolvens was identified by modified secA1 sequencing. Antibiotic treatment and removal of the central venous catheter resolved the infection. Inappropriate management of the catheter during an overnight stay outside of the hospital was considered as a possible source of infection. CONCLUSIONS: SecA1 sequencing may be a useful diagnostic tool in the identification of T. tyrosinosolvens. Providing proper central venous catheter care instructions to patients, their families, and medical staff is important for infection prevention.

Focusing on Gordonia Infections: Distribution, Antimicrobial Susceptibilities and Phylogeny.

The immunosuppression conditions and the presence of medical devices in patients favor the Gordonia infections. However, the features of this aerobic actinomycete have been little explored. Strains (n = 164) were characterized with 16S rDNA and secA1 genes to define their phylogenetic relationships, and subjected to broth microdilution to profile the antimicrobial susceptibilities of Gordonia species that caused infections in Spain during the 2005-2021 period. Four out of the eleven identified species were responsible for 86.0% of the infections: Gordonia sputi (53.0%), Gordonia bronchialis (18.3%), Gordonia terrae (8.5%) and Gordonia otitidis (6.1%). Respiratory tract infections (61.6%) and bacteremia (21.9%) were the most common infections. The secA1 gene resolved the inconclusive identification, and two major clonal lineages were observed for G. sputi and G. bronchialis. Species showed a wide antimicrobial susceptibility profile. Cefoxitin resistance varies depending on the species, reaching 94.2% for G. sputi and 36.0% for G. terrae. What is noteworthy is the minocycline resistance in G. sputi (11.5%), the clarithromycin resistance in G. bronchialis secA1 lineage II (30.0%) and the amoxicillin-clavulanate and cefepime resistance in G. terrae (21.4% and 42.8%, respectively). G. sputi and G. bronchialis stand out as the prevalent species causing infections in Spain. Resistance against cefoxitin and other antimicrobials should be considered.

Protein secretion zones during overexpression of amylase within the Gram-positive cell wall.

BACKGROUND: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis. RESULTS: We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system. CONCLUSIONS: Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus.

Síntomas óticos y síndrome del dolor miofascial en musculatura temporomandibular y esternocleidomastoideo: Estudio de un caso / Otic symptoms and myofascial pain syndrome in temporomandibular and sternocleidomastoid musculature: A case study

Introducción El dolor de oído y la alteración de la audición son motivos de consulta muy frecuentes. El dolor de oído referido puede deberse a alteraciones temporomandibulares y puntos gatillo en la musculatura cervical. La hipoacusia se relaciona con la presencia de puntos gatillo en musculatura cervical y mandibular. Aunque se han observado efectos beneficiosos sobre esta musculatura tras la utilización de terapia manual y punción seca, la literatura al respecto sigue siendo escasa, motivo por que se realizó el actual estudio. Objetivo Conocer los efectos de un tratamiento basado en la técnica suiza y punción seca en la musculatura temporomandibular y esternocleidomastoideo a corto plazo sobre el dolor de oído e hipoacusia, en una paciente de 24 años. Material y métodos Estudio de un caso. Se realizó una valoración previa al tratamiento y otra al finalizar para valorar sus efectos, mediante audiometría y escala visual analógica. Se programó una sesión de 50 min, semanalmente, durante cuatro semanas. Durante el tratamiento se aplicó la técnica suiza y punción seca en cinco músculos de la región mandibular y cervical. Resultados Tras cuatro sesiones de tratamiento se observó una mejora del dolor de la paciente y se evidenció una recuperación de la audición. Conclusiones Una intervención basada en la técnica suiza y punción seca para el tratamiento del síndrome de dolor miofascial de la musculatura temporomandibular y esternocleidomastoideo puede ser útil para mejorar alteraciones de la audición. Se sugiere la realización de ensayos clínicos aleatorizados que evalúen esta técnica (AU)

Introduction Ear pain and hearing impairment is a frequent medical consultation. Referred ear pain may be due to temporomandibular disorders and the presence of trigger points in the cervical musculature. Hearing loss is related to the presence of trigger points in the cervical and mandibular muscles. Although beneficial effects have been observed on this musculature after the use of manual therapy and dry needling, the literature on the matter is still scarce, which is why the current study was carried out. Objective To know the short-term effects of a treatment based on the Swiss technique and dry needling on the temporomandibular and sternocleidomastoid muscles on ear pain and hearing loss, in a female 24-year-old patient. Material and methods Case study. An assessment was carried out before the treatment and another at the end to evaluate its effects. A 50-minute session was held weekly for 4 weeks. During treatment, the Swiss technique was applied to 5 muscles of the mandibular and cervical region. Results After 4 treatment sessions an improvement in the patient's pain was observed and a hearing recovery was evidenced. Conclusions An intervention based on the Swiss technique and dry needling in the treatment of myofascial pain syndrome of the temporomandibular and sternocleidomastoid muscles may be useful in improving hearing disorders and otalgia. Randomized clinical trials evaluating this technique are suggested (AU)

Humic-like Substances (HULIS) in Ship Engine Emissions: Molecular Composition Effected by Fuel Type, Engine Mode, and Wet Scrubber Usage.

Humic-like substances (HULIS), known for their substantial impact on the atmosphere, are identified in marine diesel engine emissions obtained from five different fuels at two engine loads simulating real world scenarios as well as the application of wet sulfur scrubbers. The HULIS chemical composition is characterized by electrospray ionization (ESI) ultrahigh resolution mass spectrometry and shown to contain partially oxidized alkylated polycyclic aromatic compounds as well as partially oxidized aliphatic compounds, both including abundant nitrogen- and sulfur-containing species, and clearly different to HULIS emitted from biomass burning. Fuel properties such as sulfur content and aromaticity as well as the fuel combustion efficiency and engine mode are reflected in the observed HULIS composition. When the marine diesel engine is operated below the optimum engine settings, e.g., during maneuvering in harbors, HULIS-C emission factors are increased (262-893 mg kg-1), and a higher number of HULIS with a shift toward lower degree of oxidation and higher aromaticity is detected. Additionally, more aromatic and aliphatic CHOS compounds in HULIS were detected, especially for high-sulfur fuel combustion. The application of wet sulfur scrubbers decreased the HULIS-C emission factors by 4-49% but also led to the formation of new HULIS compounds. Overall, our results suggest the consideration of marine diesel engines as a relevant regional source of HULIS emissions.

A unifying mechanism for protein transport through the core bacterial Sec machinery.

Encapsulation and compartmentalization are fundamental to the evolution of cellular life, but they also pose a challenge: how to partition the molecules that perform biological functions-the proteins-across impermeable barriers into sub-cellular organelles, and to the outside. The solution lies in the evolution of specialized machines, translocons, found in every biological membrane, which act both as gate and gatekeeper across and into membrane bilayers. Understanding how these translocons operate at the molecular level has been a long-standing ambition of cell biology, and one that is approaching its denouement; particularly in the case of the ubiquitous Sec system. In this review, we highlight the fruits of recent game-changing technical innovations in structural biology, biophysics and biochemistry to present a largely complete mechanism for the bacterial version of the core Sec machinery. We discuss the merits of our model over alternative proposals and identify the remaining open questions. The template laid out by the study of the Sec system will be of immense value for probing the many other translocons found in diverse biological membranes, towards the ultimate goal of altering or impeding their functions for pharmaceutical or biotechnological purposes.

Comparison of strip meniscometry and Schirmer tear test results and tear film breakup time between healthy dogs and dogs with dry eye disease / Comparação entre o teste de meniscometria em tira, o teste lacrimal de Schirmer e o tempo de ruptura do filme lacrimal em cães normais e com olho seco

ABSTRACT Purpose: To compare strip meniscometry and Schirmer tear test 1 results and tear film breakup time between dogs with normal eyes and dogs diagnosed with keratoconjunctivitis sicca. Methods: One-hundred fifty-six eyes of 78 dogs, 88 normal eyes, and 68 eyes diagnosed with keratoconjunctivitis sicca were included in the study. The tests were performed in the following sequence: Schirmer tear test 1 was used to allocate the dogs to the normal or keratoconjunctivitis sicca group, followed by the strip meniscometry test and tear film breakup time measurement. Results: The results (mean ± standard deviation) of the tests in the normal group were as follows: Schirmer tear test 1, 22.75 ± 3.88 mm/min; strip meniscometry test, 10.01 ± 2.35 mm/5 sec; and tear film breakup time, 25.82 ± 5.47 sec. In the keratoconjunctivitis sicca group, the results were as follows: Schirmer tear test 1, 6.10 ± 4.44 mm/min; strip meniscometry test, 3.03 ± 2.62 mm/5 sec; and tear film breakup time, 10.78 ± 4.23 sec. The Spearman correlation coefficient in the keratoconjunctivitis sicca group was very high, with a significant difference between the strip meniscometry test and Schirmer tear test 1 (r=0.848, p<0.001), and moderate and significant between the strip meniscometry test and tear film breakup time (r=0.773, p<0.001). The cutoff for the strip meniscometry test for keratoconjunctivitis sicca was 7.0 mm/5 sec. Keratoconjunctivitis sicca was suspected when the values were <10 mm/5 sec. Conclusions: This study provided strip meniscometry test values from dogs with normal eyes and eyes with keratoconjunctivitis sicca, with high sensitivity and specificity compared with those of the Schirmer tear test 1. In the future, the strip meniscometry test may be another important quantitative test and could complement the gold standard Schirmer tear test for the diagnosis of keratoconjunctivitis sicca in dogs, an excellent animal model for the study of the disease.

RESUMO Objetivo: Comparar o teste de meniscometria em tira com o teste lacrimal de Schirmer 1 e o tempo de ruptura do filme lacrimal, em cães com olhos normais e cães com diagnóstico de ceratoconjuntivite seca. Métodos: Cento e cinquenta e seis olhos de 78 cães, 88 olhos normais e 68 olhos com diagnóstico de ceratoconjuntivite seca. Os testes foram realizados na seguinte sequência: o teste lacrimal de Schirmer 1 foi utilizado para alocar os cães no grupo normal ou no grupo ceratoconjuntivite seca, seguido pelo teste de meniscometria em tira e tempo de ruptura do filme lacrimal. Resultados: As médias e desvios-padrão para os olhos normais foram: teste lacrimal de Schirmer 1 = 22,75 ± 3,88 mm / min; teste de meniscometria em tira = 10,01 ± 2,35 mm / 5 seg; tempo de ruptura do filme lacrimal = 25,82 ± 5,47 seg; e para os olhos do grupo ceratoconjuntivite seca foram: teste lacrimal de Schirmer 1 = 6,10 ± 4,44 mm / min; teste de meniscometria em tira = 3,03 ± 2,62 mm / 5 seg; tempo de ruptura do filme lacrimal = 10,78 ± 4,23 seg. O teste de correlação de Spearman no grupo ceratoconjuntivite seca foi muito alto, com diferença significativa entre teste de meniscometria em tira e teste lacrimal de Schirmer 1 (r=0,848, p<0,001), moderada e significativa entre teste de meniscometria em tira e tempo de ruptura do filme lacrimal (r=0,773, p<0,001). O cut-off para teste de meniscometria em tira para ceratoconjuntivite seca foi identificado em 7,0 mm / 5 seg, valores abaixo de 10 mm / 5 seg podem ser considerados suspeitos para KCS. Conclusões: Este estudo forneceu valores de teste de meniscometria em tira em olhos normais e com ceratoconjuntivite seca em cães, revelando alta sensibilidade e especificidade em comparação com o teste lacrimal de Schirmer 1. No futuro, o teste de meniscometria em tira pode ser outro teste quantitativo importante e pode complementar o teste lacrimal de Schirmer padrão ouro para o diagnóstico de ceratoconjuntivite seca em cães, um excelente modelo animal para o estudo de ceratoconjuntivite seca.

First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with faba bean phyllody symptoms in India.

Vicia faba L. commonly known as broad bean or faba bean is one of the most widely grown protein rich legume crops. Out of more than 50 faba bean-producing countries, about 90% production is concentrated in the Asian, European Union (EU), and African region (FAO, 2020). Owing to its high nutritional value, both the fresh pods and dry seeds are consumed. During March 2022, some plants with little leaf and phyllody symptoms such as leaf-like floral structures were observed in the experimental fields of Indian Agricultural Research Institute (IARI), New Delhi (Fig. 1 a, b, c). The twig samples were collected from two individual symptomatic and one from asymptomatic plant. DNA was extracted using CTAB (cetyl trimethyl ammonium bromide) method (Ahrens and Seemüller, 1992; Marzachi et al. 1998) and examined for the association of phytoplasma through nested PCR using the universal primers P1/P7 and R16F2n/R16R2 targeting the 16SrRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and the other set of primers secAfor1/secArev3 and secAfor2/secArev3 targeting secA gene (Hodgetts et al. 2008). The DNA from symptomatic plants resulted the amplicons of 1200bp and 840bp specific to 16S rRNA and secA gene respectively. The gel purified PCR products were cloned into pGEM®-T Easy Vector system (Promega) and outsourced for Sanger sequencing at Agri Genome Labs, Kerala, India. The resultant 16S rRNA sequences (GenBank Acc. No. OP978231, OP978232) and secA sequences (ON715392 and ON715393) were examined through NCBI BLASTn analysis. The 16S rRNA sequences of the V. faba strains shared a minimum of 99.85% similarity with the phytoplasma strain causing little leaf and phyllody disease of sesame in India (MW622017) and a maximum of 100% sequence identity with the Vigna radiata phyllody and necrosis phytoplasma strain of Jodhpur (OP935760) India, whereas the secA gene sequences showed 100% identity with Tephrosia purpurea witches'-broom phytoplasma (MW603929) from China and a minimum of 91.14% similarity with 'Candidatus Phytoplasma aurantifolia' (MW020541) from India. The pairwise comparison results were completely in support of the corresponding phylogenetic sequence analysis results of 16SrRNA and secA gene sequences of faba bean strains in comparison with other strains retrieved from GenBank database, wherein the faba bean strains got clustered with 16SrII-D subgroup related strains (Fig. 2 a and b). Virtual RFLP analysis through iPhyClassifer tool through in silico digestion of R16F2n/R2 region of 16S rRNA gene of the faba bean strain using 17 restriction endonuclease enzymes resulted in the RFLP profiles similar to that of the profile of phytoplasma subgroup 16SrII-D (Y10097: papaya yellow crinkle) used as reference strain with a similarity coefficient value of 1.0. All the results of this investigation confirmed the association of 'Candidatus phytoplasma aurantifolia' (16SrII-D) with the diseased faba bean plants in this study. Previous reports of phytoplasma infecting faba bean include a group 16SrIII strain detected in Spain in 2004 (Castro and Romero), a subgroup 16SrII-D strain detected in Sudan in 2012 (Alfaro-Fernandez et al.), a group 16SrII strain detected in Saudi Arabia in 2014 (Al-Saleh and Amer), and subgroup 16SrIII-J strains detected in Egypt in 2014 (Hamed et al.) and in Peru in 2021 (Torres-Suarez et al.). To the best of our knowledge, these findings, document the first report of the association of 'Candidatus Phytoplasma aurantifolia' (subgroup 16SrII-D) with faba bean plants in India. This report necessitates further research on the status of distribution of this phytoplasma strain in other locations and hosts in the country so as to develop possible strategies to contain its further spread and management of the disease.

Comparison of the effects of six-week time-restricted eating on weight loss, body composition, and visceral fat in overweight older men and women.

The aim of this study was to determine the effectiveness of a six-week time-restricted eating (TRE) intervention in reducing body weight, fat loss, and visceral fat in overweight, older adult men and women (age range = 65-74 years). Another objective was to determine the feasibility of widespread use of TRE in older women and men. The study randomly assigned 116 healthy, non-smoking participants to one of two conditions: TRE or educational control participants. Participants in the TRE group were instructed to not consume calorie containing beverages or food for 16 h per day, from 8:00 pm to 12:00 pm. Participants in the control group were instructed to follow a meal plan based on their previous habits. The changes in body weight and body composition were determined using a SECA mBCA 515 analyzer. The six-week TRE intervention resulted in a significant decrease in body weight in both men and women (-1.8 kg and-1.3 kg, respectively; p = 0.03). In men, a significant decrease in visceral fat mass (-0.54 l; p ≤ 0.001) and waist circumference (-2.9 cm; p ≤ 0.015) was observed. No significant changes in either visceral fat or waist circumference were observed in women. Additionally, no change in skeletal muscle mass was observed in either the control or TRE group. More than 99 % of female and 98 % of male participants were able to adhere to the prescribed time-restricted eating (16/8) plan, suggesting that this dietary approach could have beneficial effects on the body composition of overweight older men and may also reduce body weight in overweight, older women.

Structural basis of SecA-mediated protein translocation.

Secretory proteins are cotranslationally or posttranslationally translocated across lipid membranes via a protein-conducting channel named SecY in prokaryotes and Sec61 in eukaryotes. The vast majority of secretory proteins in bacteria are driven through the channel posttranslationally by SecA, a highly conserved ATPase. How a polypeptide chain is moved by SecA through the SecY channel is poorly understood. Here, we report electron cryomicroscopy structures of the active SecA-SecY translocon with a polypeptide substrate. The substrate is captured in different translocation states when clamped by SecA with different nucleotides. Upon binding of an ATP analog, SecA undergoes global conformational changes to push the polypeptide substrate toward the channel in a way similar to how the RecA-like helicases translocate their nucleic acid substrates. The movements of the polypeptide substrates in the SecA-SecY translocon share a similar structural basis to those in the ribosome-SecY complex during cotranslational translocation.

Uncovering the membrane-integrated SecAN protein that plays a key role in translocating nascent outer membrane proteins.

A large number of nascent polypeptides have to get across a membrane in targeting to the proper subcellular locations. The SecYEG protein complex, a homolog of the Sec61 complex in eukaryotic cells, has been viewed as the common translocon at the inner membrane for targeting proteins to three extracytoplasmic locations in Gram-negative bacteria, despite the lack of direct verification in living cells. Here, via unnatural amino acid-mediated protein-protein interaction analyses in living cells, in combination with genetic studies, we unveiled a hitherto unreported SecAN protein that seems to be directly involved in translocationg nascent outer membrane proteins across the plasma membrane; it consists of the N-terminal 375 residues of the SecA protein and exists as a membrane-integrated homooligomer. Our new findings place multiple previous observations related to bacterial protein targeting in proper biochemical and evolutionary contexts.

A comprehensive overview of miRNA targeting drought stress resistance in plants / Uma visão geral abrangente do miRNA visando a resistência ao estresse hídrico em plantas

MicroRNAs (miRNAs) are essential nonprotein-coding genes. In a range of organisms, miRNAs has been reported to play an essential role in regulating gene expressions at post-transcriptional level. They participate in most of the stress responsive processes in plants. Drought is an ultimate abiotic stress that affects the crop production. Therefore understanding drought stress responses are essential to improve the production of agricultural crops. Throughout evolution, plants have developed their own defense systems to cope with the adversities of environmental stresses. Among defensive mechanisms include the regulations of gene expression by miRNAs. Drought stress regulates the expression of some of the functionally conserved miRNAs in different plants. The given properties of miRNAs provide an insight to genetic alterations and enhancing drought resistance in cereal crops. The current review gives a summary to regulatory mechanisms in plants as well as miRNAs response to drought stresses in cereal crops. Some possible approaches and guidelines for the exploitation of drought stress miRNA responses to improve cereal crops are also described.

MicroRNAs (miRNAs) são genes essenciais não codificadores de proteínas. Em uma variedade de organismos, foi relatado que miRNAs desempenham papel essencial na regulação da expressão gênica em nível pós-transcricional. Eles participam da maioria dos processos responsivos ao estresse nas plantas. A seca é um estresse abiótico final que afeta a produção agrícola. Portanto, compreender as respostas ao estresse da seca é essencial para melhorar a produção de safras agrícolas. Ao longo da evolução, as plantas desenvolveram seus próprios sistemas de defesa para lidar com as adversidades do estresse ambiental. Entre os mecanismos de defesa está a regulação da expressão gênica por miRNAs. O estresse hídrico regula a expressão de alguns dos miRNAs funcionalmente conservados em diferentes plantas. As propriedades dadas dos miRNAs fornecem uma visão das alterações genéticas e aumentam a resistência à seca nas safras de cereais. A revisão atual apresenta um resumo dos mecanismos regulatórios nas plantas, bem como a resposta dos miRNAs ao estresse hídrico nas plantações de cereais. Algumas abordagens e diretrizes possíveis para a exploração das respostas do miRNA ao estresse da seca para melhorar as safras de cereais também são descritas.

A comprehensive overview of miRNA targeting drought stress resistance in plants

Abstract MicroRNAs (miRNAs) are essential nonprotein-coding genes. In a range of organisms, miRNAs has been reported to play an essential role in regulating gene expressions at post-transcriptional level. They participate in most of the stress responsive processes in plants. Drought is an ultimate abiotic stress that affects the crop production. Therefore understanding drought stress responses are essential to improve the production of agricultural crops. Throughout evolution, plants have developed their own defense systems to cope with the adversities of environmental stresses. Among defensive mechanisms include the regulations of gene expression by miRNAs. Drought stress regulates the expression of some of the functionally conserved miRNAs in different plants. The given properties of miRNAs provide an insight to genetic alterations and enhancing drought resistance in cereal crops. The current review gives a summary to regulatory mechanisms in plants as well as miRNAs response to drought stresses in cereal crops. Some possible approaches and guidelines for the exploitation of drought stress miRNA responses to improve cereal crops are also described.

Resumo MicroRNAs (miRNAs) são genes essenciais não codificadores de proteínas. Em uma variedade de organismos, foi relatado que miRNAs desempenham papel essencial na regulação da expressão gênica em nível pós-transcricional. Eles participam da maioria dos processos responsivos ao estresse nas plantas. A seca é um estresse abiótico final que afeta a produção agrícola. Portanto, compreender as respostas ao estresse da seca é essencial para melhorar a produção de safras agrícolas. Ao longo da evolução, as plantas desenvolveram seus próprios sistemas de defesa para lidar com as adversidades do estresse ambiental. Entre os mecanismos de defesa está a regulação da expressão gênica por miRNAs. O estresse hídrico regula a expressão de alguns dos miRNAs funcionalmente conservados em diferentes plantas. As propriedades dadas dos miRNAs fornecem uma visão das alterações genéticas e aumentam a resistência à seca nas safras de cereais. A revisão atual apresenta um resumo dos mecanismos regulatórios nas plantas, bem como a resposta dos miRNAs ao estresse hídrico nas plantações de cereais. Algumas abordagens e diretrizes possíveis para a exploração das respostas do miRNA ao estresse da seca para melhorar as safras de cereais também são descritas.

Growth and functional leaf traits of coppice regrowth of Bertholletia excelsa during an El Niño event in the central Amazon / Crescimento e características funcionais foliares de rebrotas de Bertholletia excelsa durante um evento de El Niño na Amazônia Central

The most severe drought of this century in the Amazon rainforest, which was caused by El Niño, occurred from 2015 to 2016. With a focus on the ecophysiology of the regrowth of the Brazil nut tree, Bertholletia excelsa, it was investigated how the progression of the drought of 2015-2016 affected the physiological traits of the coppice regrowth of B. excelsa. The experiment was carried out in a ten-year-old plantation of Brazil nut trees, which had been subjected to thinning and coppice regrowth two years earlier. In the sprouts grown on the stumps of cut trees, the following treatments were applied: (T1) thinning to one sprout per stump; (T2) thinning to two sprouts per stump, and (T3) maintenance of three sprouts per stump. Thinning treatments did not alter the growth and ecophysiological traits of the Brazil nut tree sprouts, though the phosphorus content of the leaves was higher in T1. However, the progression of the drought in 2015-2016 negatively affected the growth (height) and gas exchange of sprouts of all treatments. In addition, an increase of around 37% was observed in the intrinsic wateruse efficiency. Concerning photochemical performance, no alterations were observed. Therefore, drought stress promoted a negative effect on sprout growth and affected traits related to the photosynthesis of the B. excelsa sprouts independently of the number of sprouts per stump.(AU)

A seca mais severa deste século na floresta amazônica, causada por El Niño, ocorreu de 2015 a 2016. Com foco na ecofisiologia da rebrota da castanheira da Amazônia, foi investigado como a progressão da seca de 2015-2016 afetou as características fisiológicas das rebrotas de uma talhadia de B. excelsa. O experimento foi realizado em uma plantação de castanheiras com dez anos, a qual havia sido submetida a um desbaste e rebrota de talhadia dois anos antes. Nas rebrotas crescidas sobre os tocos das árvores cortadas foram aplicados os seguintes tratamentos: (T1) desbrota para manter um broto por cepa; (T2) desbrota para manter dois brotos por cepa; e (T3) manutenção de três brotos por cepa. Os tratamentos de desbrota não alteraram o crescimento e as características ecofisiológicas dos brotos da castanheira, exceto para o teor foliar de fósforo, que foi maior em T1. Porém, a progressão da seca em 2015-2016 afetou negativamente o crescimento em altura e as trocas gasosas dos brotos de todos os tratamentos. Além disso, foi observado um aumento de cerca de 37% na eficiência intrínseca do uso da água. Quanto ao desempenho fotoquímico, não foram observadas alterações. Portanto, o estresse hídrico promoveu efeito negativo no crescimento da brotação e afetou características relacionadas à fotossíntese das brotações de B. excelsa, independentemente do número de brotações por cepa.(AU)

A comprehensive overview of miRNA targeting drought stress resistance in plants / Uma visão geral abrangente do miRNA visando a resistência ao estresse hídrico em plantas

Abstract MicroRNAs (miRNAs) are essential nonprotein-coding genes. In a range of organisms, miRNAs has been reported to play an essential role in regulating gene expressions at post-transcriptional level. They participate in most of the stress responsive processes in plants. Drought is an ultimate abiotic stress that affects the crop production. Therefore understanding drought stress responses are essential to improve the production of agricultural crops. Throughout evolution, plants have developed their own defense systems to cope with the adversities of environmental stresses. Among defensive mechanisms include the regulations of gene expression by miRNAs. Drought stress regulates the expression of some of the functionally conserved miRNAs in different plants. The given properties of miRNAs provide an insight to genetic alterations and enhancing drought resistance in cereal crops. The current review gives a summary to regulatory mechanisms in plants as well as miRNAs response to drought stresses in cereal crops. Some possible approaches and guidelines for the exploitation of drought stress miRNA responses to improve cereal crops are also described.

Resumo MicroRNAs (miRNAs) são genes essenciais não codificadores de proteínas. Em uma variedade de organismos, foi relatado que miRNAs desempenham papel essencial na regulação da expressão gênica em nível pós-transcricional. Eles participam da maioria dos processos responsivos ao estresse nas plantas. A seca é um estresse abiótico final que afeta a produção agrícola. Portanto, compreender as respostas ao estresse da seca é essencial para melhorar a produção de safras agrícolas. Ao longo da evolução, as plantas desenvolveram seus próprios sistemas de defesa para lidar com as adversidades do estresse ambiental. Entre os mecanismos de defesa está a regulação da expressão gênica por miRNAs. O estresse hídrico regula a expressão de alguns dos miRNAs funcionalmente conservados em diferentes plantas. As propriedades dadas dos miRNAs fornecem uma visão das alterações genéticas e aumentam a resistência à seca nas safras de cereais. A revisão atual apresenta um resumo dos mecanismos regulatórios nas plantas, bem como a resposta dos miRNAs ao estresse hídrico nas plantações de cereais. Algumas abordagens e diretrizes possíveis para a exploração das respostas do miRNA ao estresse da seca para melhorar as safras de cereais também são descritas.

Social wasps (Vespidae: Polistinae) in Cerrado and Caatinga conservation units, Minas Gerais, Brazil / Vespas sociais (Vespidae: Polistinae) em unidades de conservação do Cerrado e da Caatinga, Minas Gerais, Brasil

Abstract The Parque Nacional Grande Sertão Veredas, northern Minas Gerais, is located in the Cerrado, a global hotspot, and the literature lacks information about its communities of social wasps, insects that perform numerous services for the functioning of ecosystems. Therefore, this study aimed to conduct an inventory of social wasps (Vespidae: Polistinae) in the Parque Nacional Grande Sertão Veredas, and compare its richness and composition with three other fully protected conservation units in northern Minas Gerais and Vale do Jequitinhonha, Minas Gerais. A total of 41 species of 14 genera of social wasps were recorded, including a new occurrence in the state: Mischocyttarus campestris Raw, 1985. The presence of distinct social wasp communities in the Cerrado and Caatinga conservation units analyzed in this study, reinforces and justifies the creation of these fully protected areas, which ensure the conservation of this biological heritage and the environmental services provided by these taxa, besides providing subsidies for the management and sustainable use of the natural resources of these conservation units.

Resumen O Parque Nacional Grande Sertão Veredas, norte de Minas Gerais, inserido no Cerrado, hotspot mundial, carece de informações sobre a comunidade de vespas sociais, insetos que desempenham inúmeros serviços para o funcionamento dos ecossistemas. Portanto, o objetivo deste estudo foi realizar um inventário de vespas sociais (Vespidae: Polistinae) no Parque Nacional Grande Sertão Veredas, além de comparar sua riqueza e composição com à de outras três Unidades de Conservação de proteção integral no Norte e no Vale do Jequitinhonha no estado de Minas Gerais. Foram registradas 41 espécies de 14 gêneros de vespas sociais, com um novo registro de ocorrência para o estado, Mischocyttarus campestris Raw, 1985. A presença de comunidades distintas de vespas sociais nas Unidades de Conservação no Cerrado e Caatinga, analisadas no presente estudo, reforça e justifica a criação dessas áreas de proteção integral, que assegura a conservação desse patrimônio biológico, bem como os serviços ambientais prestados por esses táxons, além de fornecer subsídios para o manejo e uso sustentável dos recursos naturais dessas Unidades de Conservação.