Whole-genome sequencing of Crimean-Congo hemorrhagic fever virus circulating in Pakistan during 2022.

Pakistan is an endemic country for Crimean-Congo hemorrhagic fever (CCHF) and its Balochistan province is considered a hotspot region for circulation of the virus whereas sporadic cases have been reported from other parts of the country. Our study aims to investigate the genomic diversity of the CCHF virus circulating in Punjab and Khyber Pakhtunkhwa provinces of Pakistan. Between April to September 2022, 46 samples from suspected CCHF patients were tested, with 6 (13%) showing positive RT-PCR results. Among the positive cases, all were male, aged 14-48 years among which 4 were butchers. Three CCHF patients succumbed to the disease. The complete S-M-L-gene fragments of 4 positive samples were sequenced. The S and L segments belonged to the Asia-1 clade and clustered with regional strains from Iran, India, and Afghanistan. One M segment sequence grouped into Africa-2 along with those reported from India during 2016-2019. We report the detection of a reassorted virus (NIH-PAK-CCHF-03|2022) having Asia-1-Africa-2-Asia-1 (S-M-L) segment pattern for the first time from Pakistan. Mutational analysis showed M segments harboring eight mutations (T55A, S80P, T110I, T185A, T189A, A212T, and N239I/T) in the mucin-like domain, five mutations (D250N, T333S, I375V, M401I, A433T), four mutations (N545D, Y657F, K688R, and I824V) in GP38-domain, and three mutations (T1418N, A1431V, and G1449S) in Gc-domain. These findings highlight the significance of whole-genome sequencing of indigenous strains for a better understanding of the CCHFV evolution in Pakistan.

Genomic characterization of the first mpox and varicella-zoster co-infection in Pakistan (2023) through next-generation sequencing.

The global mpox outbreak spanning 2022-2023 has affected numerous countries worldwide. In this study, we present the first report on the detection, whole-genome sequence, and coinfection of the mpox virus and varicella zoster virus (VZV) from Pakistan. During April-May 2023, samples from 20 suspected cases of mpox were tested at the National Institutes of Health, Islamabad among which 4 tested positive. All four cases had a travel history of Saudi Arabia. All the suspected samples were processed by using a Zymo research kit for DNA extraction, followed by qRT-PCR amplification by using a DaAn Gene detection kit for the mpox virus. Further, two of the positive samples with a low Ct value (<20) were subjected to whole-genome sequencing using a metagenomic approach on the iSeq (Illumina) platform. The sequencing results revealed Clade IIb and genotype A.2.1 of MPXV, which clustered with viruses from Slovenia and the UK in July and June 2022, respectively. Our analysis identified two novel nonsynonymous substitutions in mpox virus, namely V98I in OPG046 and P600S in OPG109. Furthermore, we successfully retrieved the complete genome of VZV from the same sample, belonging to Clade 5. This study represents the first positive case of MPXV in Pakistan and the coinfection of mpox and VZV by using a metagenome approach providing insights into their complete genomes. Our results highlight the importance of surveillance at the point of entries, strengthening lab capacities including next-generation sequencing, and using differential diagnosis for timely and accurate detection of mpox cases.

Genomic diversity of SARS-CoV-2 in Pakistan during the fourth wave of pandemic.

The emergence of different variants of concern of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in upsurges of coronavirus disease 2019 (COVID-19) cases around the globe. Pakistan faced the fourth wave of COVID-19 from July to August 2021 with 314,786 cases. To understand the genomic diversity of circulating SARS-CoV-2 strains during the fourth wave of the pandemic in Pakistan, this study was conducted. The samples from 140 COVID-19-positive patients were subjected to whole-genome sequencing using the iSeq Sequencer by Illumina. The results showed that 97% (n = 136) of isolates belonged to the delta variant while three isolates belonged to alpha and only one isolate belonged to the beta variant. Among delta variant cases, 20.5% (n = 28) isolates were showing B.1.617.2 while 23.5% (n = 25), 17.59% (n = 19), 14.81% (n = 16), and 13.89% (n = 15) of isolates were showing AY.108, AY.43 AY.127, and AY.125 lineages, respectively. Islamabad was found to be the most affected city with 65% (n = 89) of delta variant cases, followed by Karachi (17%, n = 23), and Rawalpindi (10%, n = 14). Apart from the characteristic spike mutations (T19R, L452R, T478K, P681R, and D950N) of the delta variant, the sublineages exhibited other spike mutations as E156del, G142D, T95I, A222V, G446V, K529N, N532S, Q613H, and V483A. The phylogenetic analysis revealed the introductions from Singapore, the United Kingdom, and Germany. This study highlights the circulation of delta variants (B.1.617.2 and sublineages) during the fourth wave of pandemic in Pakistan.

Impact of DNA source on genetic variant detection from human whole-genome sequencing data.

BACKGROUND: Whole blood is currently the most common DNA source for whole-genome sequencing (WGS), but for studies requiring non-invasive collection, self-collection, greater sample stability or additional tissue references, saliva or buccal samples may be preferred. However, the relative quality of sequencing data and accuracy of genetic variant detection from blood-derived, saliva-derived and buccal-derived DNA need to be thoroughly investigated. METHODS: Matched blood, saliva and buccal samples from four unrelated individuals were used to compare sequencing metrics and variant-detection accuracy among these DNA sources. RESULTS: We observed significant differences among DNA sources for sequencing quality metrics such as percentage of reads aligned and mean read depth (p<0.05). Differences were negligible in the accuracy of detecting short insertions and deletions; however, the false positive rate for single nucleotide variation detection was slightly higher in some saliva and buccal samples. The sensitivity of copy number variant (CNV) detection was up to 25% higher in blood samples, depending on CNV size and type, and appeared to be worse in saliva and buccal samples with high bacterial concentration. We also show that methylation-based enrichment for eukaryotic DNA in saliva and buccal samples increased alignment rates but also reduced read-depth uniformity, hampering CNV detection. CONCLUSION: For WGS, we recommend using DNA extracted from blood rather than saliva or buccal swabs; if saliva or buccal samples are used, we recommend against using methylation-based eukaryotic DNA enrichment. All data used in this study are available for further open-science investigation.

AI and Ethics: A Systematic Review of the Ethical Considerations of Large Language Model Use in Surgery Research.

INTRODUCTION: As large language models receive greater attention in medical research, the investigation of ethical considerations is warranted. This review aims to explore surgery literature to identify ethical concerns surrounding these artificial intelligence models and evaluate how autonomy, beneficence, nonmaleficence, and justice are represented within these ethical discussions to provide insights in order to guide further research and practice. METHODS: A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Five electronic databases were searched in October 2023. Eligible studies included surgery-related articles that focused on large language models and contained adequate ethical discussion. Study details, including specialty and ethical concerns, were collected. RESULTS: The literature search yielded 1179 articles, with 53 meeting the inclusion criteria. Plastic surgery, orthopedic surgery, and neurosurgery were the most represented surgical specialties. Autonomy was the most explicitly cited ethical principle. The most frequently discussed ethical concern was accuracy (n = 45, 84.9%), followed by bias, patient confidentiality, and responsibility. CONCLUSION: The ethical implications of using large language models in surgery are complex and evolving. The integration of these models into surgery necessitates continuous ethical discourse to ensure responsible and ethical use, balancing technological advancement with human dignity and safety.

Role of Silicon Counteracting Cadmium Toxicity in Alfalfa (Medicago sativa L.).

Cadmium (Cd) is one of the most phytotoxic elements causing an agricultural problem and human health hazards. This work investigates whether and how silicon (Si) ameliorates Cd toxicity in Alfalfa. The addition of Si in Cd-stressed plants caused significant improvement in morpho-physiological features as well as total protein and membrane stability, indicating that Si does have critical roles in Cd detoxification in Alfalfa. Furthermore, Si supplementation in Cd-stressed plants showed a significant decrease in Cd and Fe concentrations in both roots and shoots compared with Cd-stressed plants, revealing that Si-mediated tolerance to Cd stress is associated with Cd inhibition in Alfalfa. Results also showed no significant changes in the expression of two metal chelators [MsPCS1 (phytochelatin synthase) and MsMT2 (metallothionein)] and PC (phytochelatin) accumulation, indicating that there may be no metal sequestration or change in metal sequestration following Si application under Cd stress in Alfalfa. We further performed a targeted study on the effect of Si on Fe uptake mechanisms. We observed the consistent reduction in Fe reductase activity, expression of Fe-related genes [MsIRT1 (Fe transporter), MsNramp1 (metal transporter) and OsFRO1 (ferric chelate reductase] and Fe chelators (citrate and malate) by Si application to Cd stress in roots of Alfalfa. These results support that limiting Fe uptake through the down-regulation of Fe acquisition mechanisms confers Si-mediated alleviation of Cd toxicity in Alfalfa. Finally, an increase of catalase, ascorbate peroxidase, and superoxide dismutase activities along with elevated methionine and proline subjected to Si application might play roles, at least in part, to reduce H2O2 and to provide antioxidant defense against Cd stress in Alfalfa. The study shows evidence of the effect of Si on alleviating Cd toxicity in Alfalfa and can be further extended for phytoremediation of Cd toxicity in plants.

Regulation of Phytosiderophore Release and Antioxidant Defense in Roots Driven by Shoot-Based Auxin Signaling Confers Tolerance to Excess Iron in Wheat.

Iron (Fe) is essential but harmful for plants at toxic level. However, how wheat plants tolerate excess Fe remains vague. This study aims at elucidating the mechanisms underlying tolerance to excess Fe in wheat. Higher Fe concentration caused morpho-physiological retardation in BR 26 (sensitive) but not in BR 27 (tolerant). Phytosiderophore and 2-deoxymugineic acid showed no changes in BR 27 but significantly increased in BR 26 due to excess Fe. Further, expression of TaSAMS. TaDMAS1, and TaYSL15 significantly downregulated in BR 27 roots, while these were upregulated in BR 26 under excess Fe. It confirms that inhibition of phytosiderophore directs less Fe accumulation in BR 27. However, phytochelatin and expression of TaPCS1 and TaMT1 showed no significant induction in response to excess Fe. Furthermore, excess Fe showed increased catalase, peroxidase, and glutathione reductase activities along with glutathione, cysteine, and proline accumulation in roots in BR 27. Interestingly, BR 27 self-grafts and plants having BR 26 rootstock attached to BR 27 scion had no Fe-toxicity induced adverse effect on morphology but showed BR 27 type expressions, confirming that shoot-derived signal triggering Fe-toxicity tolerance in roots. Finally, auxin inhibitor applied with higher Fe concentration caused a significant decline in morpho-physiological parameters along with increased TaSAMS and TaDMAS1 expression in roots of BR 27, revealing the involvement of auxin signaling in response to excess Fe. These findings propose that tolerance to excess Fe in wheat is attributed to the regulation of phytosiderophore limiting Fe acquisition along with increased antioxidant defense in roots driven by shoot-derived auxin signaling.

Reduction in Tumor Volume by Cone Beam Computed Tomography Predicts Overall Survival in Non-Small Cell Lung Cancer Treated With Chemoradiation Therapy.

PURPOSE: We sought to evaluate whether tumor response using cone beam computed tomography (CBCT) performed as part of the routine care during chemoradiation therapy (CRT) could forecast the outcome of unresectable, locally advanced, non-small cell lung cancer (NSCLC). METHODS AND MATERIALS: We manually delineated primary tumor volumes (TV) of patients with NSCLC who were treated with radical CRT on days 1, 8, 15, 22, 29, 36, and 43 on CBCTs obtained as part of the standard radiation treatment course. Percentage reductions in TV were calculated and then correlated to survival and pattern of recurrence using Cox proportional hazard models. Clinical information including histologic subtype was also considered in the study of such associations. RESULTS: We evaluated 38 patients with a median follow-up time of 23.4 months. The median TV reduction was 39.3% (range, 7.3%-69.3%) from day 1 (D1) to day 43 (D43) CBCTs. Overall survival was associated with TV reduction from D1 to D43 (hazard ratio [HR] 0.557, 95% CI 0.39-0.79, P=.0009). For every 10% decrease in TV from D1 to D43, the risk of death decreased by 44.3%. For patients whose TV decreased ≥39.3 or <39.3%, log-rank test demonstrated a separation in survival (P=.02), with median survivals of 31 months versus 10 months, respectively. Neither local recurrence (HR 0.791, 95% CI 0.51-1.23, P=.29), nor distant recurrence (HR 0.78, 95% CI 0.57-1.08, P=.137) correlated with TV decrease from D1 to D43. Histologic subtype showed no impact on our findings. CONCLUSIONS: TV reduction as determined by CBCT during CRT as part of routine care predicts post-CRT survival. Such knowledge may justify intensification of RT or application of additional therapies. Assessment of genomic characteristics of these tumors may permit a better understanding of behavior or prediction of therapeutic outcomes.

Evaluation of unnatural cyclic amino acids as boron delivery agents for treatment of melanomas and gliomas.

Unnatural cyclic amino acids (UNAAs) are a new class of boron delivery agents that are in a pre-clinical stage of evaluation. In the present study, the biodistribution of racemic forms of the cis- and trans-isomers of the boronated UNAA 1-amino-3-boronocyclopentanecarboxylic acid (ABCPC) and 1-amino-3-boronocycloheptanecarboxylic acid (ABCHC) were evaluted in B16 melanoma bearing mice and this was compared to l-p-boronophenylalanine (BPA). Boron concentrations were determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES) at 2.5h following intraperitoneal (i.p.) injection of the test agents at a concentration equivalent to 24mg/B/kg. While all compounds attained comparable tumor boron concentrations, the tumor/blood (T/Bl) boron concentration ratios were far superior for both cis-ABCPC and cis-ABCHC compared to BPA (T/Bl=16.4, and 15.1 vs. 5.4). Secondary ion mass spectrometry (SIMS) imaging revealed that the cis-ABCPC delivered boron to the nuclei, as well as the cytoplasm of B16 cells. Next, a biodistribution study of cis-ABCPC and BPA was carried out in F98 glioma bearing rats following i.p. administration. Both compounds attained comparable tumor boron concentrations but the tumor/brain (T/Br) boron ratio was superior for cis-ABCPC compared to BPA (6 vs. 3.3). Since UNAAs are water soluble and cannot be metabolized by tumor cells, they could be potentially more effective boron delivery agents than BPA. Our data suggest that further studies are warranted to evaluate these compounds prior to the initiation of clinical studies.

Effects of l-DOPA pre-loading on the uptake of boronophenylalanine using the F98 glioma and B16 melanoma models.

The present study was undertaken to evaluate the effects of l-DOPA pre-loading on the uptake of BPA using the F98 rat glioma and the murine B16 melanoma models. In vitro pretreatments of F98 glioma and B16 melanoma cells with l-DOPA, followed by exposure to BPA increased boron uptake, as determined by inductively coupled plasma-optical emission spectroscopy (ICP-OES). Based on this, in vivo studies were initiated in F98 glioma bearing rats. Initially, the l-DOPA dosing paradigm was evaluated. Maximum tumor boron uptake was observed following i.p. administration of l-DOPA (50mg/kg) followed 24h later by BPA (31.8±8.9 vs. 17.2±6.3µg/g for BPA alone). Next, the effect of l-DOPA pre-loading as a function of the route of administration of BPA was evaluated in F98 glioma bearing rats. The greatest increase in uptake was seen following i.v. administration of BPA, while in contrast no significant increase was seen following intracarotid (i.c.) administration (38.6±12.4 vs. 34.2±10.9). Cellular localization of the F98 glioma, as determined by secondary ion mass spectrometry (SIMS) boron imaging revealed equivalent tumor boron concentrations following l-DOPA pre-loading. In vivo studies in B16 melanoma bearing mice showed equivalent tumor boron values in treated and untreated mice, suggesting that the effects of l-DOPA pre-loading may depend both on the histologic type of tumor and its anatomic site.

Biodistribution and subcellular localization of an unnatural boron-containing amino acid (cis-ABCPC) by imaging secondary ion mass spectrometry for neutron capture therapy of melanomas and gliomas.

The development of new boron-delivery agents is a high priority for improving the effectiveness of boron neutron capture therapy. In the present study, 1-amino-3-borono-cyclopentanecarboxylic acid (cis-ABCPC) as a mixture of its L- and D-enantiomers was evaluated in vivo using the B16 melanoma model for the human tumor and the F98 rat glioma as a model for human gliomas. A secondary ion mass spectrometry (SIMS) based imaging instrument, CAMECA IMS 3F SIMS Ion Microscope, was used for quantitative imaging of boron at 500 nm spatial resolution. Both in vivo and in vitro studies in melanoma models demonstrated that boron was localized in the cytoplasm and nuclei with some cell-to-cell variability. Uptake of cis-ABCPC in B16 cells was time dependent with a 7.5:1 partitioning ratio of boron between cell nuclei and the nutrient medium after 4 hrs. incubation. Furthermore, cis-ABCPC delivered boron to cells in all phases of the cell cycle, including S-phase. In vivo SIMS studies using the F98 rat glioma model revealed an 8:1 boron partitioning ratio between the main tumor mass and normal brain tissue with a 5:1 ratio between infiltrating tumor cells and contiguous normal brain. Since cis-ABCPC is water soluble and can cross the blood-brain-barrier via the L-type amino acid transporters (LAT), it may accumulate preferentially in infiltrating tumor cells in normal brain due to up-regulation of LAT in high grade gliomas. Once trapped inside the tumor cell, cis-ABCPC cannot be metabolized and remains either in a free pool or bound to cell matrix components. The significant improvement in boron uptake by both the main tumor mass and infiltrating tumor cells compared to those reported in animal and clinical studies of p-boronophenylalanine strongly suggest that cis-ABCPC has the potential to become a novel new boron delivery agent for neutron capture therapy of gliomas and melanomas.