HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for accurate interpretation of in vitro data. However, variations in HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than 4-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.

Pakistan is one of the few countries where poliovirus transmission still persists, despite intensive efforts to eradicate the disease. Adequate vaccination coverage is essential to achieve polio eradication, but misconceptions about polio vaccines have hindered vaccination efforts. To address this issue, we conducted a mixed-methods study to explore knowledge and perceptions regarding polio disease and immunization in high-risk areas of Pakistan. We collected quantitative data from 3780, 1258, and 2100 households in Karachi, Bajaur, and Pishin, respectively, and supplemented this with qualitative data from focus group discussions and in-depth interviews. Our findings reveal a high level of awareness about polio and its immunization; however, misperceptions about the polio vaccine persist, leading to vaccine refusal for both polio and routine immunization. Our study provides up-to-date data on knowledge and perceptions of polio and its immunization and identifies critical gaps. These findings can inform the development of future strategies and innovative approaches to improve the success of the polio program in Pakistan.

Fruit production is an important part of the gross domestic product for many countries around the world especially to those who have a strong focus on agriculture. However, long-term maintenance and yield stability of fruit production may be threatened by the ongoing climate change and its consequences like extended drought periods, heavy rain events, and floodings. Genome editing, with its progressive technological developments, offers opportunities to adapt relevant fruit plant species to new climatic conditions. Among modern genome editing techniques, CRISPR/Cas, in particular, has the potential to support breeding for those fruit plant species with extended breeding cycles, e.g., perennial fruits. In this review, we discuss CRISPR/Cas and other genome editing techniques in detail and how these techniques can be applied to support the breeding of fruit plant species for adaptation to changing climates. The chronological history of CRISPR/Cas9 systems, their associated computational tools, genomic data sources, transformation methods along with their delivery vehicles, quality improvement, environmental-stress resiliency, limitations, and future perspectives will also be discussed with respect to securing future global fruit production.

The OPV is the vaccine of choice in polio eradication, especially in developing countries, as it has eliminated the wild poliovirus type 2. However, the immunity induced by IPV is better than that induced by the OPV. The present study compared the mucosal and humoral response to poliovirus vaccines administered to previously OPV-immunized children to assess the immunity gap in children at-risk of high poliovirus transmission. This was a community-based three-arm cluster randomized controlled trial conducted from June 2013 to May 2014 in healthy children under five years of age living in three high-risk districts of Pakistan, i.e., Karachi, Kashmore, and Bajaur. 387 clusters were randomized (131 to arm A, 127 to arm B, and 129 to arm C); however, 360 remained in the trial until the end (116 in arm A, 122 in arm B, and 122 in arm C). These clusters were randomly allocated using a computer algorithm to receive routine polio program (bOPV) activities (control, arm A), additional interventions with community mobilization and provision of short-term preventive maternal and child health services and routine immunization, including bOPV via health camps, (arm B), or all interventions of arm B with an additional provision of IPV (arm C ~ bOPV and IPV). Blood and stool samples were collected from a sub-sample to estimate humoral and intestinal immunity. Study findings showed that the serum titers were highest in Group C (IPV+OPV) at the baseline for P1, where its increase over time was also more prominent. Titers for P2 and P3 were statistically significantly higher amongst those who had received a routine OPV dose versus those who had not; this was true for all study groups and visits. In populations with high Oral Polio Vaccine (OPV) failure rates, administering an Inactivated Polio Vaccine (IPV) booster after a minimum of two OPV doses may effectively bridge immunity gaps. The IPV alone offers limited benefits to humoral immunity and doesn't provide intestinal immunity to prevent the infection and propagation of live poliovirus among unexposed populations.

Implants are used to replace damaged biological structures in human body. Although stain-less-steel (SS) is a well-known implant material, corrosion of SS implants led to the release toxic metallic ions which produce harmful effects in human body. To prevent material degradation and its harmful repercussions, these implanted materials are subjected to biocompatible coatings. Polymeric coatings play a vital role in enhancing the mechanical and biological integrity of the implanted devices. Zein is a natural protein extracted from corn and is known to have good bio-compatibility and biodegradability. In this study, Zein/Ag-Sr doped mesoporous bioactive glass nanoparticles (Ag-Sr MBGNs) were deposited on SS substrates via electrophoretic deposition (EPD) at different parameters. Ag and Sr ions were added to impart antibacterial and osteogenic properties to the coatings, respectively. In order to examine the surface morphology of coatings, optical microscopy and scanning electron microscopy (SEM) were performed. To analyze me-chanical strength, pencil scratch test, bend test, corrosion and wear tests were conducted on ze-in/Ag-Sr doped MBGNs coatings. The results show good adhesion strength, wettability, corro-sion, and wear resistance for zein/Ag-Sr doped MBGNs coatings as compared to bare SS sub-strate. Thus, good mechanical and biological properties were observed for zein/Ag-Sr doped MBGNs coatings. Results suggested these zein/Ag-Sr MBGNs coatings have great potential in bone regeneration applications.

Crop plants should be resilient to climatic factors in order to feed ever-increasing populations. Plants have developed stress-responsive mechanisms by changing their metabolic pathways and switching the stress-responsive genes. The discovery of plant transcriptional factors (TFs) as key regulators of different biotic and abiotic stresses have opened up new horizons for plant scientists. TFs perceive the signal and switch certain stress-responsive genes on and off by binding to different cis-regulatory elements. The above 50 species of plant TFs have been reported in nature. DREB, bZIP, MYB, NAC, Zinc-finger, HSF, Dof, WRKY, and NF-Y are important with respect to biotic and abiotic stresses whereas the role of many TFs is yet to explore. In this review, we summarize the role of different stress-responsive TFs with respect to biotic and abiotic stresses. Further, challenges and future opportunities linked with TFs for developing climate-resilient crops are also elaborated.

Hydrogels can provide instant relief to pain and facilitate the fast recovery of wounds. Currently the incorporation of medicinal herbs/plants in polymer matrix is being investigated due to their anti-bacterial and wound healing properties. Herein, we investigated the novel combination of chitosan (CS), chondroitin sulfate (CHI) and garlic (Gar) to synthesize hydrogels through freeze gelation process for faster wound healing and resistance to microbial growth at the wound surface. The synthesized hydrogels were characterized via Fourier transform infrared spectroscopy (FTIR), which confirmed the presence of relevant functional groups. The scanning electron microscopy (SEM) images exhibited the porous structure of the hydrogels, which is useful for the sustained release of Gar from the hydrogels. The synthesized hydrogels showed significant inhibition zones against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Furthermore, cell culture studies confirmed the cyto-compatibility of the synthesized hydrogels. Thus, the novel hydrogels presented in this study can offer antibacterial effect during wound healing and promote tissue regeneration.

Introduction: Child stunting remains a public health concern. It is characterized as poor cognitive and physical development in children due to inadequate nutrition during the first 1,000 days of life. Across South Asia, Pakistan has the second-highest prevalence of stunting. This study has assessed the most recent nationally representative data, the National Nutrition Survey (NNS) 2018, to identify the stunting prevalence and determinants among Pakistani children under five. Methods: The NNS 2018, a cross-sectional household-level survey, was used to conduct a secondary analysis. Data on malnutrition, dietary practices, and food insecurity was used to identify the prevalence of stunting among children under five years by demographic, socioeconomic, and geographic characteristics. The prevalence of stunting was calculated using the World Health Organization (WHO) height for age z-score references. Univariate and multivariable logistic regressions were conducted to identify factors associated with child stunting. Results: Analysis showed that out % of 52,602 children under five, 40.0% were found stunted. Male children living in rural areas were more susceptible to stunting. Furthermore, stunting was more prevalent among children whose mothers had no education, were between 20 and 34, and were employed. In the multivariable logistic regression, male children (AOR=1.08, 95% CI[1.04-1.14], P<0.001) from rural areas (AOR=1.07, 95% CI [1.01-1.14], P=0.014), with the presence of diarrhea in the last two weeks (AOR=1.15, 95% CI [1.06-1.25], P<0.001), with mothers who have no education (AOR=1.57, 95% CI [1.42-1.73], P<0.001) or lower levels of education (Primary: AOR=1.35, 95% CI [1.21-1.51], P<0.001; Middle: AOR=1.29, 95% CI [1.15-1.45], P<0.001) had higher odds of stunting. Younger children aged <6 months (AOR=0.53, 95% CI [0.48-0.58], P<0.001) and 6-23 months (AOR=0.89, 95% CI [0.84-0.94], P<0.001), with mothers aged 35-49 years (AOR=0.78, 95% CI [0.66-0.92], P=0.003) had lower odds of stunting. At the household level, the odds of child stunting were higher in the poorest households (AOR=1.64, 95% CI [1.46-1.83], P<0.001), with ≥7 members (AOR=1.09, 95% CI [1.04-1.15], P<0.001), with no access to improved sanitation facilities (AOR=1.14, 95% CI [1.06-1.22], P<0.001) and experiencing severe food insecurity (AOR=1.07, 95% CI [1.01-1.14], P=0.02). Conclusion: Child stunting in Pakistan is strongly associated with various factors, including gender, age, diarrhea, residence, maternal age and education, household size, food and wealth status, and access to sanitation. To address this, interventions must be introduced to make locally available food and nutritious supplements more affordable, improve access to safe water and sanitation, and promote female education for long-term reduction in stunting rates.