Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding / Isolamento e caracterização de bactérias associadas ao trato intestinal do bicho-da-seda sob alimentação larval tratada com antibióticos

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding

Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.

Regulation of autophagy by SARS-CoV-2: The multifunctional contributions of ORF3a.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) regulates autophagic flux by blocking the fusion of autophagosomes with lysosomes, causing the accumulation of membranous vesicles for replication. Multiple SARS-CoV-2 proteins regulate autophagy with significant roles attributed to ORF3a. Mechanistically, open reading frame 3a (ORF3a) forms a complex with UV radiation resistance associated, regulating the functions of the PIK3C3-1 and PIK3C3-2 lipid kinase complexes, thereby modulating autophagosome biogenesis. ORF3a sequesters VPS39 onto the late endosome/lysosome, inhibiting assembly of the soluble NSF attachement protein REceptor (SNARE) complex and preventing autolysosome formation. ORF3a promotes the interaction between BECN1 and HMGB1, inducing the assembly of PIK3CA kinases into the ER (endoplasmic reticulum) and activating reticulophagy, proinflammatory responses, and ER stress. ORF3a recruits BORCS6 and ARL8B to lysosomes, initiating the anterograde transport of the virus to the plasma membrane. ORF3a also activates the SNARE complex (STX4-SNAP23-VAMP7), inducing fusion of lysosomes with the plasma membrane for viral egress. These mechanistic details can provide multiple targets for inhibiting SARS-CoV-2 by developing host- or host-pathogen interface-based therapeutics.

Polyamines and nitric oxide crosstalk in plant development and abiotic stress tolerance.

Polyamines (PAs) and nitric oxide (NO) are crucial signalling molecules that exhibit a promising role in improving stress tolerance in plants, maintaining their growth and development. They act as protecting agents for plants through activation of stress adaptation strategies such as membrane stabilisation, acid neutralisation and suppression of ROS generation. NO interacts with PAs during several developmental processes and stress responses. External supplementation of PAs to plants is also reported to cause an increase in NO content. However, it is unclear whether PAs promote synthesis of NO by either as substrates, cofactors, or signals. Impact of NO on synthesis of PAs has been also reported in some studies, yet the exact governing mechanisms of the interrelation between NO and PAs is currently obscure. Understanding the crosstalk between PAs and NO during growth and stress condition in plants can aid in providing better tolerance to plants against stressful environment.

The exploitation of host autophagy and ubiquitin machinery by Mycobacterium tuberculosis in shaping immune responses and host defense during infection.

Intracellular pathogens have evolved various efficient molecular armaments to subvert innate defenses. Cellular ubiquitination, a normal physiological process to maintain homeostasis, is emerging one such exploited mechanism. Ubiquitin (Ub), a small protein modifier, is conjugated to diverse protein substrates to regulate many functions. Structurally diverse linkages of poly-Ub to target proteins allow enormous functional diversity with specificity being governed by evolutionarily conserved enzymes (E3-Ub ligases). The Ub-binding domain (UBD) and LC3-interacting region (LIR) are critical features of macroautophagy/autophagy receptors that recognize Ub-conjugated on protein substrates. Emerging evidence suggests that E3-Ub ligases unexpectedly protect against intracellular pathogens by tagging poly-Ub on their surfaces and targeting them to phagophores. Two E3-Ub ligases, PRKN and SMURF1, provide immunity against Mycobacterium tuberculosis (M. tb). Both enzymes conjugate K63 and K48-linked poly-Ub to M. tb for successful delivery to phagophores. Intriguingly, M. tb exploits virulence factors to effectively dampen host-directed autophagy utilizing diverse mechanisms. Autophagy receptors contain LIR-motifs that interact with conserved Atg8-family proteins to modulate phagophore biogenesis and fusion to the lysosome. Intracellular pathogens have evolved a vast repertoire of virulence effectors to subdue host-immunity via hijacking the host ubiquitination process. This review highlights the xenophagy-mediated clearance of M. tb involving host E3-Ub ligases and counter-strategy of autophagy inhibition by M. tb using virulence factors. The role of Ub-binding receptors and their mode of autophagy regulation is also explained. We also discuss the co-opting and utilization of the host Ub system by M. tb for its survival and virulence.Abbreviations: APC: anaphase promoting complex/cyclosome; ATG5: autophagy related 5; BCG: bacille Calmette-Guerin; C2: Ca2+-binding motif; CALCOCO2: calcium binding and coiled-coil domain 2; CUE: coupling of ubiquitin conjugation to ER degradation domains; DUB: deubiquitinating enzyme; GABARAP: GABA type A receptor-associated protein; HECT: homologous to the E6-AP carboxyl terminus; IBR: in-between-ring fingers; IFN: interferon; IL1B: interleukin 1 beta; KEAP1: kelch like ECH associated protein 1; LAMP1: lysosomal associated membrane protein 1; LGALS: galectin; LIR: LC3-interacting region; MAPK11/p38: mitogen-activated protein kinase 11; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAPK8/JNK: mitogen-activated protein kinase 8; MHC-II: major histocompatibility complex-II; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB1/p50: nuclear factor kappa B subunit 1; OPTN: optineurin; PB1: phox and bem 1; PE/PPE: proline-glutamic acid/proline-proline-glutamic acid; PknG: serine/threonine-protein kinase PknG; PRKN: parkin RBR E3 ubiquitin protein ligase; RBR: RING-in between RING; RING: really interesting new gene; RNF166: RING finger protein 166; ROS: reactive oxygen species; SMURF1: SMAD specific E3 ubiquitin protein ligase 1; SQSTM1: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; Ub: ubiquitin; UBA: ubiquitin-associated; UBAN: ubiquitin-binding domain in ABIN proteins and NEMO; UBD: ubiquitin-binding domain; UBL: ubiquitin-like; ULK1: unc-51 like autophagy activating kinase 1.

Heat stress develops with increased total-tract gut permeability, and dietary organic acid and pure botanical supplementation partly restores lactation performance in Holstein dairy cows.

To evaluate the effects of heat stress (HS) conditions and dietary organic acid and pure botanical (OA/PB) supplementation on gut permeability and milk production, we enrolled 46 multiparous Holstein cows [208 ± 4.65 dry matter intake (DMI; mean ± SD), 3.0 ± 0.42 lactation, 122 ± 4.92 d pregnant, and 39.2 ± 0.26 kg of milk yield] in a study with a completely randomized design. Cows were assigned to 1 of 4 groups: thermoneutral conditions (TN-Con, n = 12), HS conditions (HS-Con, n = 12), thermoneutral conditions pair-fed to HS-Con (TN-PF, n = 12), or HS supplemented with OA/PB [75 mg/kg of body weight (BW); 25% citric acid, 16.7% sorbic acid, 1.7% thymol, 1.0% vanillin, and 55.6% triglyceride; HS-OAPB, n = 10]. Supplements were delivered twice daily by top-dress; all cows not supplemented with OA/PB received an equivalent amount of the triglyceride used for microencapsulation of the OA/PB supplement as a top-dress. Cows were maintained in thermoneutrality [temperature-humidity index (THI) = 68] during a 7-d acclimation and covariate period. Thereafter, cows remained in thermoneutral conditions or were moved to HS conditions (THI: diurnal change 74 to 82) for 14 d. Cows were milked twice daily. Clinical assessments and BW were recorded, blood was sampled, and gastrointestinal permeability measurements were repeatedly evaluated. The mixed model included fixed effects of treatment, time, and their interaction. Rectal and skin temperatures and respiration rates were greater in HS-Con and HS-OAPB relative to TN-Con. Dry matter intake, water intake, and yields of energy-corrected milk (ECM), protein, and lactose were lower in HS-Con relative to HS-OAPB. Nitrogen efficiency was improved in HS-OAPB relative to HS-Con. Compared with TN-Con and TN-PF, milk yield and ECM were lower in HS-Con cows. Total-tract gastrointestinal permeability measured at d 3 of treatment was greater in HS-Con relative to TN-Con or TN-PF. Plasma total fatty acid concentrations were reduced, whereas insulin concentrations were increased in HS-Con relative to TN-PF. We conclude that exposure to a heat-stress environment increases total-tract gastrointestinal permeability. This study highlights important mechanisms that might account for milk production losses caused by heat stress, independent of changes in DMI. Our observations also suggest that dietary supplementation of OA/PB is a means to partly restore ECM production and improve nitrogen efficiency in dairy cattle experiencing heat stress.

Can Mycobacterium tuberculosis infection lead to cancer? Call for a paradigm shift in understanding TB and cancer.

Infections are known to cause tumours though more attributed to viruses. Strong epidemiological links suggest association between bacterial infections and cancers as exemplified by Helicobacter pylori and Salmonella spp. Infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), has been reported to predispose patients to lung cancers and possibly in other organs as well. While this etiopathogenesis warrant inclusion of M. tb in IARC's (International Agency for Research on Cancer) classified carcinogenic agents, the lack of well-defined literature and direct experimental studies have barred the research community from accepting the role of M. tb as a carcinogen. The background research, case studies, and experimental data extensively reviewed in Roy et al., 2021; provoke the debate for elucidating carcinogenic properties of M. tb. Moreover, proper, timely and correct diagnosis of both diseases (which often mimic each other) will save millions of lives that are misdiagnosed. In addition, use of Anti Tubercular therapy (ATT) in misdiagnosed non-TB patients contributes to drug resistance in population thereby severely impacting TB disease control measures. Research in this arena can further aid in saving billions of dollars by preventing the superfluous use of cancer drugs. In order to achieve these goals, it is imperative to identify the underlying mechanism of M. tb infection acting as major risk factor for cancer.

COVID-19 and tuberculosis: the double whammy of respiratory pathogens.

Prior to coronavirus disease 2019 (COVID-19), tuberculosis (TB) was the worst killer among infectious diseases. The union of these two obnoxious respiratory diseases can be devastating, with severe public health implications. The COVID-19 pandemic has affected all TB-elimination programmes due to the severe burden on healthcare systems and the diversion of funds and attention towards controlling the pandemic. The emerging data show that the COVID-19 pandemic caused a marked decrease in case notifications and bacille Calmette-Guérin immunisations, ultimately promoting disease transmission and increasing the susceptible population. The similarity between the clinical characteristics of TB and COVID-19 adds to the public health complications, with evidence of immune dysregulation in both cases leading to severe consequences. Clinical evidence suggests that severe acute respiratory syndrome coronavirus 2 infection predisposes patients to TB infection or may lead to reactivation of latent disease. Similarly, underlying TB disease can worsen COVID-19. Treatment options are limited in COVID-19; therefore, using immunosuppressive and immunomodulatory regimens that can modulate the concomitant bacterial infection and interaction with anti-TB drugs requires caution. Thus, considering the synergistic impact of these two respiratory diseases, it is crucial to manage both diseases to combat the syndemic of TB and COVID-19.

SVIoT: A Secure Visual-IoT Framework for Smart Healthcare.

The advancement of the Internet of Things (IoT) has transfigured the overlay of the physical world by superimposing digital information in various sectors, including smart cities, industry, healthcare, etc. Among the various shared information, visual data are an insensible part of smart cities, especially in healthcare. As a result, visual-IoT research is gathering momentum. In visual-IoT, visual sensors, such as cameras, collect critical multimedia information about industries, healthcare, shopping, autonomous vehicles, crowd management, etc. In healthcare, patient-related data are captured and then transmitted via insecure transmission lines. The security of this data are of paramount importance. Besides the fact that visual data requires a large bandwidth, the gap between communication and computation is an additional challenge for visual IoT system development. In this paper, we present SVIoT, a Secure Visual-IoT framework, which addresses the issues of both data security and resource constraints in IoT-based healthcare. This was achieved by proposing a novel reversible data hiding (RDH) scheme based on One Dimensional Neighborhood Mean Interpolation (ODNMI). The use of ODNMI reduces the computational complexity and storage/bandwidth requirements by 50 percent. We upscaled the original image from M × N to M ± 2N, dissimilar to conventional interpolation methods, wherein images are upscaled to 2M × 2N. We made use of an innovative mechanism, Left Data Shifting (LDS), before embedding data in the cover image. Before embedding the data, we encrypted it using an AES-128 encryption algorithm to offer additional security. The use of LDS ensures better perceptual quality at a relatively high payload. We achieved an average PSNR of 43 dB for a payload of 1.5 bpp (bits per pixel). In addition, we embedded a fragile watermark in the cover image to ensure authentication of the received content.

Isolation and characterization of bacteria associated with silkworm gut under antibiotic-treated larval feeding.

The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.

Mycobacterium tuberculosis Protein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival.

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis (M. smegmatis) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host-pathogen interaction during M. tb infection.

Development and Validation of Signature Sequence-Based PCR for Improved Molecular Diagnosis of Tuberculosis.

Reliable, fast, and affordable diagnosis for tuberculosis (TB) remains a challenge to reduce disease incidence in resource-poor countries. Tests based on nucleotide sequences that are signature to Mycobacterium tuberculosis have the potential to make a positive impact on case detection rates, which can eventually help control TB. Using extensive comparative bioinformatics approach, we mined the genome for M. tuberculosis-specific genes and identified four genes so-called signature sequence (SS). With <25% homology with other known genes/proteins of mycobacterial/nonmycobacterial origin in various databases, these SS genes are ideal targets for species-specific identification. Sputum from suspected patients was liquefied using novel complete liquefying reagent, and DNA was isolated. Samples from patients (n = 417), reporting to TB clinics at two different hospitals, which met our inclusion criteria, were collected for this study. A small number (n = 143) was used for initial standardization, and the remaining patient samples (n = 274) were evaluated by SS and compared with smear microscopy, GeneXpert, culture, and clinical outcome. An overwhelming sensitivity of 97.0%, significantly higher than GeneXpert (95.0%), was seen. SS could pick all smear-negative, but culture-positive samples, along with other culture-negative samples; some of the latter were declared clinically positive. Our results yielded superior sensitivity and specificity through conventional PCR.

Violence against Healthcare Workers during the COVID-19 Pandemic: A Review of Incidents from a Lower-Middle-Income Country.

Background: Healthcare workers (HCWs) across the globe have met tremendous challenges during the COVID-19 (coronavirus disease 2019) pandemic, such as shortages of personal protective equipment, extensive work hours, and constant fear of catching the virus or transmitting it to loved ones. Adding on to the already existing burnout, an increase in incidents of violence and aggression against HCWs was seen in Pakistan and globally. Objectives: Primarily to review cases of violence against HCWs in Pakistan, highlighting and comparing the instigating factors seen within the country and globally. Secondly, to enlist possible interventions to counter workplace violence in healthcare during a pandemic and in general. Methods: Incidents of violence towards HCWs in Pakistan during the COVID-19 pandemic occurring between April 7, 2020, and August 7, 2020, were included. The incidents reported from local newspapers were reviewed. Findings and Conclusion: A total of 29 incidents were identified, with perpetrators of violence most commonly being relatives of COVID-19 patients. Most frequent reasons included mistrust in HCWs, belief in conspiracy theories, hospitals' refusal to admit COVID-19 patients due to limited space, COVID-19 hospital policies, and the death of the COVID-19 patients. Protests by doctors and other HCWs for provision of adequate PPE, better quarantine conditions for doctors with suspected COVID-19, and better compensation for doctors on COVID-19 patient duty resulted in police violence towards HCWs. To avoid such incidents in the future, institutions, healthcare policymakers, media organisations, and law enforcement agencies must work together for widespread public awareness to counter misconceptions and to exhibit responsible journalism. In hospitals, measures such as de-escalation training and increased security must be implemented. Furthermore, law enforcement agencies must be trained in non-violent methods of crowd dispersal and control to manage peaceful protests by HCWs over legitimate issues.

Prevalence and risk factors of Syphilis among blood donors of Punjab, Pakistan.

Syphilis is a sexually transmitted disease and its actual prevalence among Pakistani blood donors is currently unknown. A cross sectional study was conducted at different district healthcare hospitals of Punjab, Pakistan with an aim to evaluate the prevalence and risk factors associated with syphilis in blood donors using immunochromatographic test (ICT) and enzyme linked immunosorbent assay (ELISA). A total (n=1200) blood samples were collected from donors aged 18-65 years. All the information regarding personal data, demographic data and risk factors was collected via structured questionnaire. On the basis of ICT and ELISA, the overall prevalence of syphilis was 3.91% among blood donors. The demographic factors positively linked with syphilis were age (P= 0.000; Odds ratio, OR= 7.18; 95% confidence interval CI= 2.816-18.295) and education status (P= 0.000; Odds ratio, OR= 12.33; 95% confidence interval CI= 3.469-43.849) of donors. Similarly among the risk factors analyzed, marital status (P= 0.012; Odds ratio OR= 2.251; 95% confidence interval CI= 1.206- 4.202) and blood transfusion history (P= 0.030; Odds ratio OR= 1.981; 95% confidence interval CI= 1.083-3.623) were also strongly associated with syphilis. We emphasized the importance of promoting preventive measures for syphilis. The syphilis diagnosis should not be based on a single test. The present study indicates that higher prevalence is alarming for blood donors in Pakistan. Stringent donor screening is highly recommended to ensure maximum safe blood transfusion.

Insights into decontamination of soils by phytoremediation: A detailed account on heavy metal toxicity and mitigation strategies.

In the current era of rapid industrialization, the foremost challenge is the management of industrial wastes. Activities such as mining and industrialization spill over a large quantity of toxic waste that pollutes soil, water, and air. This poses a major environmental and health challenge. The toxic heavy metals present in the soil and water are entering the food chain, which in turn causes severe health hazards. Environmental clean-up and reclamation of heavy metal contaminated soil and water are very important, and it necessitates efforts of environmentalists, industrialists, scientists, and policymakers. Phytoremediation is a plant-based approach to remediate heavy metal/organic pollutant contaminated soil and water in an eco-friendly, cost-effective, and permanent way. This review covers the effect of heavy metal toxicity on plant growth and physiological process, the concept of heavy metal accumulation, detoxification, and the mechanisms of tolerance in plants. Based on plants' ability to uptake heavy metals and metabolize them within tissues, phytoremediation techniques have been classified into six types: phytoextraction, phytoimmobilization, phytovolatilization, phytodegradation, rhizofiltration, and rhizodegradation. The development of research in this area led to the identification of metal hyper-accumulators, which could be utilized for reclamation of contaminated soil through phytomining. Concurrently, breeding and biotechnological approaches can enhance the remediation efficiency. Phytoremediation technology, combined with other reclamation technologies/practices, can provide clean soil and water to the ecosystem.

Mycobacterium tuberculosis RipA Dampens TLR4-Mediated Host Protective Response Using a Multi-Pronged Approach Involving Autophagy, Apoptosis, Metabolic Repurposing, and Immune Modulation.

Reductive evolution has endowed Mycobacterium tuberculosis (M. tb) with moonlighting in protein functions. We demonstrate that RipA (Rv1477), a peptidoglycan hydrolase, activates the NFκB signaling pathway and elicits the production of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12, through the activation of an innate immune-receptor, toll-like receptor (TLR)4. RipA also induces an enhanced expression of macrophage activation markers MHC-II, CD80, and CD86, suggestive of M1 polarization. RipA harbors LC3 (Microtubule-associated protein 1A/1B-light chain 3) motifs known to be involved in autophagy regulation and indeed alters the levels of autophagy markers LC3BII and P62/SQSTM1 (Sequestosome-1), along with an increase in the ratio of P62/Beclin1, a hallmark of autophagy inhibition. The use of pharmacological agents, rapamycin and bafilomycin A1, reveals that RipA activates PI3K-AKT-mTORC1 signaling cascade that ultimately culminates in the inhibition of autophagy initiating kinase ULK1 (Unc-51 like autophagy activating kinase). This inhibition of autophagy translates into efficient intracellular survival, within macrophages, of recombinant Mycobacterium smegmatis expressing M. tb RipA. RipA, which also localizes into mitochondria, inhibits the production of oxidative phosphorylation enzymes to promote a Warburg-like phenotype in macrophages that favors bacterial replication. Furthermore, RipA also inhibited caspase-dependent programed cell death in macrophages, thus hindering an efficient innate antibacterial response. Collectively, our results highlight the role of an endopeptidase to create a permissive replication niche in host cells by inducing the repression of autophagy and apoptosis, along with metabolic reprogramming, and pointing to the role of RipA in disease pathogenesis.

Efficient and selective catalytic hydroxylation of unsaturated plant oils: a novel method for producing anti-pathogens.

With the increasing demand for antimicrobial agents and the spread of antibiotic resistance in pathogens, the exploitation of plant oils to partly replace antibiotic emerges as an important source of fine chemicals, functional food utility and pharmaceutical industries. This work introduces a novel catalytic method of plant oils hydroxylation by Fe(III) citrate monohydrate (Fe3+-cit.)/Na2S2O8 catalyst. Methyl (9Z,12Z)-octadecadienoate (ML) was selected as an example of vegetable oils hydroxylation to its hydroxy-conjugated derivatives (CHML) in the presence of a new complex of Fe(II)-species. Methyl 9,12-di-hydroxyoctadecanoate 1, methyl-9-hydroxyoctadecanoate 2 and methyl (10E,12E)-octadecanoate 3 mixtures is produced under optimized condition with oxygen balloon. The specific hydroxylation activity was lower in the case of using Na2S2O8 alone as a catalyst. A chemical reaction has shown the main process converted of plantoils hydroxylation and (+ 16 Da) of OH- attached at the methyl linoleate (ML-OH). HPLC and MALDI-ToF-mass spectrometry were employed for determining the obtained products. It was found that adding oxidizing agents (Na2S2O8) to Fe3+ in the MeCN mixture with H2O would generate the new complex of Fe(II)-species, which improves the C-H activation. Hence, the present study demonstrated a new functional method for better usage of vegetable oils.Producing conjugated hydroxy-fatty acids/esters with better antipathogenic properties. CHML used in food industry, It has a potential pathway to food safety and packaging process with good advantages, fundamental to microbial resistance. Lastly, our findings showed that biological monitoring of CHML-minimum inhibitory concentration (MIC) inhibited growth of various gram-positive and gram-negative bacteria in vitro study. The produced CHML profiles were comparable to the corresponding to previousstudies and showed improved the inhibition efficiency over the respective kanamycin derivatives.

Effects of abomasal infusions of fatty acids and 1-carbon donors on apparent fatty acid digestibility and incorporation into milk fat in cows.

Our primary objective was to determine the effects of the abomasal infusion of 16-carbon (16C) and 22-carbon (22C) fatty acids (FA) on apparent FA digestibility, plasma FA concentrations, and their incorporation into milk fat in cows. Our secondary objective was to study the effects of 1-carbon donors choline and l-serine on these variables. Five rumen-cannulated Holstein cows (214 ± 4.9 d in milk; 3.2 ± 1.1 parity) were enrolled in a 5 × 5 Latin square experiment with experimental periods lasting 6 d. Abomasal infusates consisted of (1) palmitic acid (PA; 98% 16:0 of total fat), (2) PA + choline chloride (PA+CC; 50 g/d of choline chloride), (3) PA + l-serine (PA+S; 170 g/d of l-serine), (4) behenic acid (BA; 92% 22:0 of total fat), and (5) docosahexaenoic acid algal oil (DHA; 47.5% DHA of total fat). Emulsions were formulated to provide 301 g/d of total FA and were balanced to provide a minimum of 40 and 19 g/d of 16:0 and glycerol, respectively, to match the content found in the infused algal oil. Apparent digestibility of FA was highest in DHA, intermediate in PA, and lowest in BA. Digestibility of 16C FA was lowest in BA and highest in PA. The digestibility of 22C FA was highest in DHA relative to BA (99 vs. 58%), whereas 1-carbon donors had no effect on 22C FA digestibility. Plasma 16C FA concentrations were greatest with PA treatment, and 22C FA concentrations were ~3-fold greater in DHA-treated cows relative to all other treatments. Milk fat 16:0 content was highest in PA relative to BA and DHA (e.g., 37 vs. 27% in PA and DHA), whereas the milk yield of 16:0 was higher in PA relative to DHA (i.e., 454 vs. 235 g/d). Similarly, milk 22:0 content and yield were ~10-fold higher in BA relative to all other treatments, whereas DHA treatment resulted in higher content and yield of 22:6 in milk fat relative to all other treatments (41- and 38-fold higher, respectively). Consequently, the content of FA >16C (i.e., preformed) was higher in milk fat from cows infused with BA and DHA relative to PA. De novo FA content in milk did not differ between PA, PA+CC, and PA+S (~16% of milk fat) but was higher in BA and DHA treatments (19 and 21%, respectively). We conclude that FA carbon chain length and degree of saturation affected FA digestibility and availability for absorption as well as their incorporation into milk fat. The abomasal infusion of choline chloride and l-serine did not modify these variables relative to infusing palmitic acid alone.

Teleological cooption of Mycobacterium tuberculosis PE/PPE proteins as porins: Role in molecular immigration and emigration.

Permeation through bacterial cells for exchange or uptake of biomolecules and ions invariably depend upon the existence of pore-forming proteins (porins) in their outer membrane. Mycobacterium tuberculosis (M. tb) harbours one of the most rigid cell envelopes across bacterial genera and is devoid of the classical porins for solute transport across the cell membrane. Though canonical porins are incompatible with the evolution of permeability barrier, porin like activity has been reported from membrane preparations of pathogenic mycobacteria. This suggests a sophisticated transport mechanism that has been elusive until now, along with the protein family responsible for it. Recent evidence suggests that these slow-growing mycobacteria have co-opted some of PE/PPE family proteins as molecular transport channels, in place of porins, to facilitate uptake of nutrients required to thrive in the restrictive host environment. These reports advocate that PE/PPE proteins, due to their structural ability, have a potential role in importing small molecules to the cell's interior. This mechanism unveils how a successful pathogen overcomes its restrictive membrane's transport limitations for selective uptake of nutrients. If extrapolated to have a role in drug transport, these channels could help understand the emergence of drug resistance. Further, as these proteins are associated with the export of virulence factors, they can be exploited as novel drug targets. There remains, however, an interesting question that as the PE/PPE proteins can allow the 'import' of molecules from outside the cell, is the reverse transport also possible across the M. tb membrane. In this review, we have discussed recent evidence supporting PE/PPE's role as a specific transport channel for selective uptake of small molecule nutrients and, as possible molecular export machinery of M. tb. This newly discovered role as transmembrane channels demands further research on this enigmatic family of proteins to comprehend the pathomechanism of this very smart pathogen.

The Importance of Nitric Oxide as the Molecular Basis of the Hydrogen Gas Fumigation-Induced Alleviation of Cd Stress on Ganoderma lucidum.

Whether or not hydrogen gas (H2) can reduce cadmium (Cd) toxicity in Ganoderma lucidum has remained largely unknown. Here, we report that Cd-induced growth inhibition in G. lucidum was significantly alleviated by H2 fumigation or hydrogen-rich water (HRW), evaluated by lower oxidative damage and Cd accumulation. Moreover, the amelioration effects of H2 fumigation were better than of HRW in an optimum concentration of H2 under our experimental conditions. Further results showed that H2-alleviated growth inhibition in G. lucidum was accompanied by increased nitric oxide (NO) level and nitrate reductase (NR) activity under Cd stress. On the other hand, the mitigation effects were reversed after removing endogenous NO with its scavenger cPTIO or inhibiting H2-induced NR activity with sodium tungstate. The role of NO in H2-alleviated growth inhibition under Cd stress was proved to be achieved through a restoration of redox balance, an increase in cysteine and proline contents, and a reduction in Cd accumulation. In summary, these results clearly revealed that NR-dependent NO might be involved in the H2-alleviated Cd toxicity in G. lucidum through rebuilding redox homeostasis, increasing cysteine and proline levels, and reducing Cd accumulation. These findings may open a new window for H2 application in Cd-stressed economically important fungi.