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Abstract In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

Abstract In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

ABSTRACT The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to β-lactam antibiotics, these strains can easily acquire reduced β-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced β-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA.IMPORTANCEThe emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to β-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced β-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to β-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Non-menstrual toxic shock syndrome (non-mTSS) is a life-threatening disease caused by Staphylococcus aureus strains producing superantigens, such as staphylococcal enterotoxins A, B, C, and toxic shock syndrome toxin-1 (TSST-1). However, little is known about why the TSS cases are rare, although S. aureus strains frequently carry a tst gene, which encodes TSST-1. To answer this question, the amount of TSST-1 produced by 541 clinical isolates was measured in both the presence and absence of serum supplementation to growth media. Then a set of S. aureus strains with similar genetic backgrounds isolated from patients presenting with non-mTSS and those with clinical manifestations other than non-mTSS was compared for their TSST-1 inducibility by human serum, and their whole-genome sequences were determined. Subsequently, the association of mutations identified in the tst promoter of non-mTSS strains with TSST-1 inducibility by human serum was evaluated by constructing promoter replacement mutants and green fluorescent protein (GFP) reporter recombinants. Results showed that 39 out of 541 clinical isolates (7.2%), including strains isolated from non-mTSS patients, had enhanced production of TSST-1 in the presence of serum. TSST-1 inducibility by human serum was more clearly seen in non-mTSS strains of clonal complex (CC)-5. Moreover, the whole-genome sequence analysis identified a set of sequence variations at a putative SarA-binding site of the tst promoter. This sequence variation was proven to be partially responsible for the induction of TSST-1 production by human serum. We conclude that the onset of staphylococcal toxic shock syndrome caused by TSST-1-producing CC-5 strains seem at least partially initiated by serum induction of TSST-1, which is regulated by the mutation of putative SarA-binding site at the tst promoter.

Evaluation of genotypes under multiple environments is the prerequisite for the development of stable and superior genotypes for sustainable barley production and a changing climate. GGE (G, genotype and GE, genotype (G) by environment (E), interaction) biplot and the AMMI (The Additive Main effects and Multiplicative Interaction) model are the effective methods to find out the genotype(s) which are stable and suitable to cultivate in specific or multiple environments. The experiment was conducted to analyze the performance of seven barley genotypes for selecting stable and superior genotypes across three different environmental conditions of Bangladesh (i.e., at the Bangladesh Agricultural Research Institute (BARI), Gazipur; at the Regional Agricultural Research Station (RARS), BARI, Jamalpur and at the RARS, BARI Ishurdi). All genotypes in three locations were arranged in a randomized complete block design (RCBD) with three replications. After two years observation, it was found that all genotypes across the location were found highly significant (p≤0.01), due to the variation of environments, genotypic variability and their interaction. The first two principle component axes (PC1 and PC2) of site regression model were significant (P≤0.01) and cumulatively contributed to 89.65% of the total GE interaction. In the polygon view of biplot, there were five rays which divided the biplot into five sectors, and all three locations fell into two of these five sections. Location Jamalpur fell into sector 1, whereas Ishurdi and Gazipur fell into sector 2. Among the locations, Ishurdi was found the best for all genotypes, where Gazipur and Jamalpur were found unfavourable. Among the genotypes, ‘E7’ performed the best for the average grain yield (GY) followed by ‘E3’, ‘E2’ and ‘E4’, whereas ‘E1’ had lowest average GY for all locations. The highest yield in environment Jamalpur was obtained by the genotype ‘E2’, on the other hand genotype ‘E7’ produced the highest GY in locations of Ishurdi and Gazipur. Considering yield stability, genotypes ‘E3’, ‘E4’ and ‘E1’ were found to be more stable, whereas genotype ‘E2’ was the most unstable over all locations. Genotypes ‘E7’ and ‘E3’ were found to be close to the ideal genotype position, in the case of the maximum GY and yield stability across the locations as compared to other genotypes and recommended for commercial cultivation for Bangladesh including South-Asia.

Dry bean (Phaseolus vulgaris L.) is an important worldwide legume crop with low to moderate levels of resistance to common bacterial blight (CBB) caused by Xanthomonas axonopodis pv. phaseoli. A total of 852 genotypes (cultivars, preliminary and advanced breeding lines) from the North Dakota State University dry bean breeding program were tested for their effectiveness as populations for genome-wide association studies (GWAS) to identify genomic regions associated with resistance to CBB, to exploit the associated markers for marker-assisted breeding (MAB), and to identify candidate genes. The genotypes were evaluated in a growth chamber for disease resistance at both the unifoliate and trifoliate stages. At the unifoliate stage, 35% of genotypes were resistant, while 25% of genotypes were resistant at the trifoliate stage. Libraries generated from each genotype were sequenced using the Illumina platform. After filtering for sequence quality, read depth, and minor allele frequency, 41,998 single-nucleotide polymorphisms (SNPs) and 30,285 SNPs were used in GWAS for the Middle American and Andean gene pools, respectively. One region near the distal end of Pv10 near the SAP6 molecular marker from the Andean gene pool explained 26.7–36.4% of the resistance variation. Three to seven regions from the Middle American gene pool contributed to 25.8–27.7% of the resistance, with the most significant peak also near the SAP6 marker. Six of the eight total regions associated with CBB resistance are likely the physical locations of quantitative trait loci identified from previous genetic studies. The two new locations associated with CBB resistance are located at Pv10:22.91–23.36 and Pv11:52.4. A lipoxgenase-1 ortholog on Pv10 emerged as a candidate gene for CBB resistance. The state of one SNP on Pv07 was associated with susceptibility. Its subsequent use in MAB would reduce the current number of lines in preliminary and advanced field yield trial by up to 14% and eliminate only susceptible genotypes. These results provide a foundational SNP data set, improve our understanding of CBB resistance in dry bean, and impact resource allocation within breeding programs as breeding populations may be used for dual purposes: cultivar development as well as genetic studies.

AimUnintended pregnancy is a significant public health concern in South Asian countries because of its negative association with the socioeconomic and health outcomes for both children and mothers. The present study aimed to explore the prevalence of unintended pregnancy and explore its determinants among women of reproductive age in six South Asian countries.MethodsNationwide latest demography and health survey data from six South Asian countries, including Bangladesh (2014), Pakistan (2017-2018), Nepal (2016), Afghanistan (2015), Maldives (2016-2017) and India (2015-2016) were pooled for the present study. Multivariate analysis was performed to explore the association between unintended pregnancy and its associated factors.ResultsAmongst the total women (n = 41,689), overall, 19.1% pregnancies were reported as unintended (ranging from 11.9% in India to 28.4% in Bangladesh). The logistic regression model showed that younger women (15-19 years) had 1.42 times higher chance of unintended pregnancies. The odds of unintended pregnancies was 1.24 times higher for poorest women and 1.19 times higher for poorer women. Further, urban women (aOR = 0.70, 95% CI = 0.50-0.80), women having no children (aOR = 0.10, 95% CI = 0.09-0.12), smaller (≤4) family (aOR = 0.72, 95% CI = 0.67-0.78), those who intent to use contraceptive (aOR = 0.72, 95% CI = 0.60-0.86), currently living with partner (aOR = 0.90, 95% CI = 0.81-0.99), first cohabitation in teenage (≤19 years) (aOR = 0.85, 95% CI = 0.78-0.92) were less likely to report unintended pregnancies.ConclusionsThis study has showed that women's age, wealth index, place of residence, number of children, family size, the intention of contraceptive use, living with a partner, and first cohabitation age are essential determinants of unintended pregnancy. These factors should be considered when trying to reduce unintended pregnancy in six South Asian countries. However, there is a need to improve health education, counselling, skills-building, sex education, modern contraceptive use and its access in this region. Intervention programs regarding reproductive health and policies are warranted to reduce rates of unintended pregnancy in South Asian countries.

In the face of sudden migration to online teaching due to Covid-19 pandemic, education is going under trying times globally, especially in the low resource contexts of the Global South. Little is, however, known about how this forced migration to and coping with emergency remote teaching (ERT) are happening in the low resource context of developing Bangladesh, particularly in the Higher Education (HE). This paper, therefore, aims to understand how virtual classrooms look like in the Higher Education context in Bangladesh through TESOL teachers' narratives from initial virtual classrooms imparting ERT. Employing narrative inquiry, four teachers from four different public and private universities in Bangladesh share their experiences of doing, undergoing and reflecting as they try to adopt, adapt and strive in imparting ERT and carrying out education in HE. Insights from these initial teacher narratives may inform higher education pedagogies, teacher development in distance education environments in Bangladesh and similar contexts in the Global South. As the paper addresses HE teachers' initial orientation to ERT without exclusively dealing with TESOL education, it has multidisciplinary approach to migration to and coping with ERT in HE in general.

Malaysia is anticipating an increase of 68.86% in CO2 emission in 2020, compared with the 2000 baseline, reaching 285.73 million tonnes. A major contributor to Malaysia’s CO2 emissions is coal-fired electricity power plants, responsible for 43.4% of the overall emissions. Malaysia’s forest soil offers organic sequestration of 15 tonnes of CO2 ha−1·year−1. Unlike organic CO2 sequestration in soil, inorganic sequestration of CO2 through mineral carbonation, once formed, is considered as a permanent sink. Inorganic CO2 sequestration in Malaysia has not been extensively studied, and the country’s potential for using the technique for atmospheric CO2 removal is undefined. In addition, Malaysia produces a significant amount of solid waste annually and, of that, demolition concrete waste, basalt quarry fine, and fly and bottom ashes are calcium-rich materials suitable for inorganic CO2 sequestration. This project introduces a potential solution for sequestering atmospheric CO2 inorganically for Malaysia. If lands associated to future developments in Malaysia are designed for inorganic CO2 sequestration using demolition concrete waste, basalt quarry fine, and fly and bottom ashes, 597,465 tonnes of CO2 can be captured annually adding a potential annual economic benefit of €4,700,000.