Novel Variant and Known Mutation in 23S rRNA Gene of Mycoplasma pneumoniae, Northern Vietnam, 2023.

During a 2023 outbreak of Mycoplasma pneumoniae-associated community-acquired pneumonia among children in northern Vietnam, we analyzed M. pneumoniae isolated from nasopharyngeal samples. In almost half (6 of 13) of samples tested, we found known A2063G mutations (macrolide resistance) and a novel C2353T variant on the 23S rRNA gene.

A newly constructed Agrobacterium-mediated transformation system based on the hisB auxotrophic marker for genetic manipulation in Aspergillus niger.

The filamentous fungus Aspergillus niger is widely exploited as an industrial workhorse for producing enzymes and organic acids. So far, different genetic tools, including CRISPR/Cas9 genome editing strategies, have been developed for the engineering of A. niger. However, these tools usually require a suitable method for gene transfer into the fungal genome, like protoplast-mediated transformation (PMT) or Agrobacterium tumefaciens-mediated transformation (ATMT). Compared to PMT, ATMT is considered more advantageous because fungal spores can be used directly for genetic transformation instead of protoplasts. Although ATMT has been applied in many filamentous fungi, it remains less effective in A. niger. In the present study, we deleted the hisB gene and established an ATMT system for A. niger based on the histidine auxotrophic mechanism. Our results revealed that the ATMT system could achieve 300 transformants per 107 fungal spores under optimal transformation conditions. The ATMT efficiency in this work is 5 - 60 times higher than those of the previous ATMT studies in A. niger. The ATMT system was successfully applied to express the DsRed fluorescent protein-encoding gene from the Discosoma coral in A. niger. Furthermore, we showed that the ATMT system was efficient for gene targeting in A. niger. The deletion efficiency of the laeA regulatory gene using hisB as a selectable marker could reach 68 - 85% in A. niger strains. The ATMT system constructed in our work represents a promising genetic tool for heterologous expression and gene targeting in the industrially important fungus A. niger.

An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens.

OBJECTIVES: This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). RESULTS: In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. CONCLUSIONS: The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

Psychological impact of the state of emergency over COVID-19 for non-permanent workers: a Nationwide follow-up study in Japan.

BACKGROUND: The outbreak of COVID-19 has caused mental health problems and increased unemployment due to the economic recession. This survey aimed to assess the psychological impact of the state of emergency. We estimated changes in mental health, quality of life, and unemployment experience for general workers during the first COVID-19 outbreak in Japan. METHODS: We conducted a nationwide follow-up study. During the periods of March 26 to April 6, 2020 and June 26 to July 2, 2020, we used the internet to survey general workers aged 15 to 59 years in Japan. The questionnaire items covered employment status and socioeconomic factors, and we used the Center for Epidemiologic Studies Depression Scale (CES-D) and EQ-5D-5L to assess depression and health-related quality of life (HR-QOL), respectively. The differences in outcomes of permanent and non-permanent workers were analyzed using propensity score analysis. A multiple linear regression analysis was performed to examine the relationship between unemployment and CES-D scores. RESULTS: We included 2351 subjects in the analysis. Changes in both CES-D scores and utility were not significantly different between the two groups. However, a significant difference was found regarding the rate of unemployment, which was associated with higher CES-D scores. CONCLUSIONS: The present study demonstrated that the mental health of non-permanent workers was not negatively affected following the state of emergency due to COVID-19 in Japan. Unemployment is an important factor that influences the mental health of general workers.

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 106 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Genome-wide identification, characterization, and expression analysis of the small auxin-up RNA gene family during zygotic and somatic embryo maturation of the cacao tree (Theobroma cacao).

Small auxin-up RNA (SAUR) proteins were known as a large family that supposedly participated in various biological processes in higher plant species. However, the SAUR family has been still not explored in cacao (Theobroma cacao L.), one of the most important industrial trees. The present work, as an in silico study, revealed comprehensive aspects of the structure, phylogeny, and expression of TcSAUR gene family in cacao. A total of 90 members of the TcSAUR gene family have been identified and annotated in the cacao genome. According to the physic-chemical features analysis, all TcSAUR proteins exhibited slightly similar characteristics. Phylogenetic analysis showed that these TcSAUR proteins could be categorized into seven distinct groups, with 10 sub-groups. Our results suggested that tandemly duplication events, segmental duplication events, and whole genome duplication events might be important in the growth of the TcSAUR gene family in cacao. By re-analyzing the available transcriptome databases, we found that a number of TcSAUR genes were exclusively expressed during the zygotic embryogenesis and somatic embryogenesis. Taken together, our study will be valuable to further functional characterizations of candidate TcSAUR genes for the genetic engineering of cacao.

Whole-genome sequencing of antimicrobial-resistant Salmonella enterica isolates from a Cairina moschata carcass.

Salmonella enterica is one of the most common agents of foodborne bacterial illness with poultry being an important reservoir. The indiscriminate use of antimicrobial compounds in poultry farming increasingly leads to antimicrobial-resistant (AMR) which threatens the health of both animals and humans. Antimicrobial-resistant Salmonella enterica from the poultry can spread to human through the direct contact with infected poultry or fecal contaminated environments. Antimicrobial-resistant S. enterica, especially fluoroquinolone-resistant nontyphoidal Salmonella is in the list of global health concern stated by the World Health Organization (WHO). Here we report the whole-genome sequencing data and de novo genome assemble of antimicrobial-resistant S. enterica strains S8 and S9 from the C. moschata carcass collected in Vietnam. Genomic DNA of S. enterica were extracted and subjected to whole-genome sequencing using Illumina MiSeq platform. The genome size of antimicrobial-resistant S. enterica strain S8 is 4,707,459 bp with a GC-content of 52.38%, containing 10 antimicrobial resistant genes. The genome size of antimicrobial-resistant Samonella enterica strain S9 is 4,923,944 bp with a GC-content of 52,39%, containing 10 antimicrobial resistance genes. Our data provided the insights on antimicrobial resistant genes of S. enterica isolates from the C. moschata carcass, which help to understand the infection mechanism of antimicrobial-resistant S. enterica in human.

A nationwide web-based survey of factors associated with depressive symptoms among Japanese workers.

BACKGROUND: Precarious employment has affected mental health, and limited data are available on the association of low stress tolerance with depressive symptoms among Japanese workers. AIMS: This study aimed to examine the relationship between stress tolerance and depressive symptoms among Japanese workers, including company employees, civil servants and self-employed persons in various industries. METHODS: We conducted a nationwide cross-sectional study. From March 26 to April 6, 2020, we performed a web-based survey of Japanese workers. The questionnaire included questions on socioeconomic factors, the SOC scale that assesses stress tolerance, the CES-D, and the EQ-5D-5L. Multivariate regression analyses were performed to determine the factors associated with depressive symptoms. RESULTS: We included 3,001 participants in the analysis. A high SOC score, adequate sleeping time and frequency of exercise were associated with higher depressive symptoms. Employment status and long working hours were not associated with depressive symptoms. Younger workers had lower SOC scores than older workers. An inverse correlation between the SOC score and CES-D score was found among Japanese workers. CONCLUSIONS: Improving stress tolerance among younger workers is needed to prevent worsening mental health regardless of employment status for Japanese workers.

Social Media Insights During the COVID-19 Pandemic: Infodemiology Study Using Big Data.

BACKGROUND: The COVID-19 pandemic is still undergoing complicated developments in Vietnam and around the world. There is a lot of information about the COVID-19 pandemic, especially on the internet where people can create and share information quickly. This can lead to an infodemic, which is a challenge every government might face in the fight against pandemics. OBJECTIVE: This study aims to understand public attention toward the pandemic (from December 2019 to November 2020) through 7 types of sources: Facebook, Instagram, YouTube, blogs, news sites, forums, and e-commerce sites. METHODS: We collected and analyzed nearly 38 million pieces of text data from the aforementioned sources via SocialHeat, a social listening (infoveillance) platform developed by YouNet Group. We described not only public attention volume trends, discussion sentiments, top sources, top posts that gained the most public attention, and hot keyword frequency but also hot keywords' co-occurrence as visualized by the VOSviewer software tool. RESULTS: In this study, we reached four main conclusions. First, based on changing discussion trends regarding the COVID-19 subject, 7 periods were identified based on events that can be aggregated into two pandemic waves in Vietnam. Second, community pages on Facebook were the source of the most engagement from the public. However, the sources with the highest average interaction efficiency per article were government sources. Third, people's attitudes when discussing the pandemic have changed from negative to positive emotions. Fourth, the type of content that attracts the most interactions from people varies from time to time. Besides that, the issue-attention cycle theory occurred not only once but four times during the COVID-19 pandemic in Vietnam. CONCLUSIONS: Our study shows that online resources can help the government quickly identify public attention to public health messages during times of crisis. We also determined the hot spots that most interested the public and public attention communication patterns, which can help the government get practical information to make more effective policy reactions to help prevent the spread of the pandemic.

Invasive Meningococcal Disease Remains a Health Threat in Vietnam People's Army.

OBJECTIVE: Despite strict surveillance, Neisseria meningitidis still causes life-threatening invasive meningococcal disease (IMD). The study aimed to describe the prevalence, clinical and subclinical features, and treatment outcomes of IMD among young soldiers of the Vietnam People's Army. METHODS: A prospective, population-based surveillance study was conducted in all Vietnamese military hospitals from January 2014 to June 2021. The presence of Neisseria meningitidis was confirmed by PCR or culture from blood or/and CSF. Epidemiological indices (incidence, serogroups, and distribution of cases by length of service), medical history, clinical and sub-clinical features, and treatment outcomes were documented and analyzed. RESULTS: There were 69 IMD cases (91% serogroup B) documented, mainly in conscripts (91%). The highest annual incidence was 3.33/100,000 soldiers per year. Of these cases, 44% were meningitis (n=30), 19% septicemia (n=13), and 38% meningococcemia (n=26). The most common clinical symptoms were neck stiffness (61 cases, 88%), petechial rash (51%), and shock (20 cases, 29%). Laboratory findings showed leukocytosis in 96% of IMD cases, PCT >0.05 (ng/mL) in 100%, elevated leukocyte count (>1,000/mm3) in 71%, and high protein >1 g/L in 70%. The overall mortality rate was 9%. Two cases were found to be resistant to ceftriaxone. Prognostic factors of severity included petechial rash (OR = 9.82, p < 0.001), septicemia (OR = 5.83, p < 0.001), meningococcemia (OR = 6.22, p < 0.001), low platelet count, prolonged prothrombin time; high PCT (AUC = 0.84, p < 0.001), and increased creatinine (AUC = 0.86, p < 0.001). CONCLUSION: IMD remains a health threat in the armed forces in Vietnam, especially among new recruits. To the best of our knowledge, this is the first study in Vietnam describing ceftriaxone resistance in Neisseria meningitidis and suggests the need to reconsider standard empiric therapy for IMD.

Transcriptome analysis of monocyte-HIV interactions.

BACKGROUND: During HIV infection and/or antiretroviral therapy (ART), monocytes and macrophages exhibit a wide range of dysfunctions which contribute significantly to HIV pathogenesis and therapy-associated complications. Nevertheless, the molecular components which contribute to these dysfunctions remain elusive. We therefore applied a parallel approach of genome-wide microarray analysis and focused gene expression profiling on monocytes from patients in different stages of HIV infection and/or ART to further characterise these dysfunctions. RESULTS: Processes involved in apoptosis, cell cycle, lipid metabolism, proteasome function, protein trafficking and transcriptional regulation were identified as areas of monocyte dysfunction during HIV infection. Individual genes potentially contributing to these monocyte dysfunctions included several novel factors. One of these is the adipocytokine NAMPT/visfatin, which we show to be capable of inhibiting HIV at an early step in its life cycle. Roughly half of all genes identified were restored to control levels under ART, while the others represented a persistent dysregulation. Additionally, several candidate biomarkers (in particular CCL1 and CYP2C19) for the development of the abacavir hypersensitivity reaction were suggested. CONCLUSIONS: Previously described areas of monocyte dysfunction during HIV infection were confirmed, and novel themes were identified. Furthermore, individual genes associated with these dysfunctions and with ART-associated disorders were pinpointed. These genes form a useful basis for further functional studies concerning the contribution of monocytes/macrophages to HIV pathogenesis. One such gene, NAMPT/visfatin, represents a possible novel restriction factor for HIV.

Improved analytical validation and pharmacokinetics of valsartan using HPLC with UV detection.

The primary objective of the study was to validate a simple and sensitive method of determining valsartan concentration in human plasma samples using high performance liquid chromatography (HPLC) combined with ultraviolet (UV) detection. Methanol appeared to be the best with a high recovery efficiency compared to other solvents such as acetonitrile, ethylacetate and methyl-tert-butyl ether. After a simple protein precipitation using methanol, the analytes were separated on a Phenomenex Luna C(18) column using 42% acetonitrile with 15 mM potassium dihydrogenphosphate in water (pH 2.0; adjusted with phosphoric acid) as the mobile phase at a flow rate of 1.2 mL/min. The standard calibration curve constructed in the concentration range of 50-2000 ng/mL showed good linearity (r(2)>0.9997). Spironolactone was used as an internal standard (IS). Valsartan and IS eluted at 10.25 and 12.17 min, respectively. The intra-day and inter-day precision and accuracy were satisfactory with relative standard deviations of less than 15%. No interference peaks or matrix effects were observed in human plasma. Valsartan concentration in human plasma was well established following a single 80 mg oral dose (Diovan capsule) to eight healthy volunteers. The current determination of valsartan concentration by protein precipitation using methanol followed by analysis using HPLC with UV detection was rapid and sensitive, and provide an alternative to the analysis of valsartan by studying its clinical applications.

The role of monocytes in the development of Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome.

BACKGROUND: Tuberculosis-associated Immune Reconstitution Inflammatory Syndrome (TB-IRIS) is a common complication of combined antiretroviral therapy (cART) in HIV-TB co-infected patients. However, the disease mechanism is poorly understood, prognosis of TB-IRIS is currently impossible, and diagnosis is highly challenging. We analyzed whether the gene expression of monocytes could be correlated with TB-IRIS pathogenesis and could be used to classify patients predisposed to TB-IRIS. METHODS: Monocyte gene expression was compared between patients who developed TB-IRIS and matched controls. We carried out whole-genome expression profiling using Affymetrix GeneChip(®) ST 1.1 arrays at two time-points: before cART initiation (baseline) and at week two post-cART initiation. For each time-point, we used different statistical approaches to identify molecular signatures which could be used as classifiers. We also functionally mapped the modulated cellular pathways using the software package Ingenuity Pathway Analysis. RESULTS: At baseline, before introduction of cART and before onset of symptoms, monocyte gene expression was already perturbed in patients who subsequently developed TB-IRIS, indicating a possible involvement of monocytes in TB-IRIS predisposition. The differences in monocyte gene expression in TB-IRIS patients became even more clear after two weeks of cART (when TB-IRIS commonly occurs), with more than 100 genes for which expression showed a fold change greater than 1.5. Both at baseline and at week two post-cART initiation, a classifier of 8 and 9 genes, respectively could be built, which allowed discrimination of TB-IRIS cases and controls. Pathway analyses revealed that the majority of the dysregulated genes in TB-IRIS - at the time of the IRIS episode, but also already at baseline - are associated with infection and inflammation. Relevant biological functions which were perturbed before/during TB-IRIS included "Role of Pattern Recognition Receptors in Recognition of Bacteria and Viruses" and "Complement System". CONCLUSION: Our results indicate an involvement of monocytes in predisposition to/development of TB-IRIS, and suggest a number of functional pathways which may play a role in TB-IRIS development. This comprehensive study of gene regulation in monocytes provides baseline data for further studies into biomarkers for prognosis and diagnosis of TB-IRIS.

Preparation and characterization of pH-independent sustained release tablet containing solid dispersion granules of a poorly water-soluble drug.

Sustained release (SR) tablets containing solid dispersions (SD) granules of a poorly water-soluble drug were prepared to investigate the controlled pH-independent release of the drug. Losartan potassium (LST), an anti-hypertensive agent was chosen as a model drug because of its pH-dependent solubility and short elimination half-life. Poloxamer 188 was used as an SD carrier. A free-flowing SD granule was prepared by adsorbing the melt of the drug and poloxamer 188 onto the surface of an adsorbent, Aerosil 300 (fumed silicon dioxide), followed by direct compression with polyethylene oxide (PEO, 5 × 10(6)) to obtain an SD-loaded SR (SD-SR) matrix tablet. Differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) revealed partially amorphous structures of the drug in the SD granules. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) images indicated adsorption of SD granules onto the surface of the adsorbent. The SD granules dissolved completely within 10 min, a dissolution rate much higher than that of pure LST. Moreover, pH-independent sustained release of LST from the SD-SR tablet was achieved for 2h in gastric fluid (pH 1.2) and for 10h in intestinal fluid (pH 6.8). A combination of SD techniques using surface adsorption and SR concepts is a promising approach to control the release rate of poorly water-soluble drugs in a pH-independent manner.

New findings on melatonin absorption and alterations by pharmaceutical excipients using the Ussing chamber technique with mounted rat gastrointestinal segments.

We examined how melatonin absorption was affected by pharmaceutical excipients using the Ussing chamber technique with mounted rat gastrointestinal (GI) segments. Melatonin absorption occurs throughout the GI tract, with the greatest absorption being in the rectum and ileum and the least in the stomach. Melatonin can be classified as a low permeability drug. P-glycoprotein (P-gp) does not affect melatonin absorption but transported rhodamine 123, a well-known P-gp substrate. The possibility of saturating P-gp by melatonin was excluded. Sodium cholate (0.5%) increased melatonin absorption, but decreased absorption at higher concentrations (1.0% and 5.0%). Sodium oleate (0.5% and 1.0%) consistently decreased melatonin absorption. Pharmaceutical excipients increased the absorption of Lucifer yellow (100 microg/mL), a paracellular probe but decreased the absorption of melatonin above the critical micelle concentration (cmc), suggesting that melatonin was transported mainly by transcellular pathway. Sodium cholate and sodium oleate, when above the cmc, resulted in micellar complexes as revealed by (1)H NMR spectra and particle size distribution. Histology tests showed mucosal damage of jejunum tissues in the presence of these excipients. The balance of tissue damage by the formation of micellar complexes could affect the melatonin absorption. This information on melatonin absorption behaviors and its modulation by pharmaceutical excipients can be used in further oral dosage formulations to affect circadian rhythm.

Modulation of microenvironmental pH and crystallinity of ionizable telmisartan using alkalizers in solid dispersions for controlled release.

The present work is an original evaluation of the microenvironmental pH (pH(M)) and crystallinity of an ionizable drug in order to enhance its dissolution using alkalizers in polyethylene glycol 6000 (PEG 6000) based solid dispersions (SDs). Telmisartan (TEL) was chosen as a model drug due to its poor and pH-dependent water solubility. The nine alkalizers used to modify the pH of TEL were MgO, NaOH, KOH, Na2CO3, NaHCO, bentonite, Na2HPO4, K2HPO4 and arginine. MgO, NaOH, KOH and Na2CO3 in the SD system significantly increased the drug dissolution rate in intestinal fluid (pH 6.8) and water. Modulation of pH(M) was clearly observed as a function of time at different fractional dimensions of tablet. Structural change in drug crystallinity to an amorphous form was also a contributing factor based on differential scanning calorimetry (DSC) thermograms and powder X-ray diffraction (PXRD) patterns. The drug frequency of the CO band decreased and the O-H broad band in the Fourier transform infrared (FTIR) spectra disappeared when these alkalizers were added. It was evident that the alkalizers in PEG 6000 based SDs synergistically enhanced dissolution of TEL not only by modulating pH(M) but also by changing drug crystallinity to an amorphous form via molecular interactions.