Genome Editing in Brassica juncea Using CRISPR/Cas9 Technology.

Modern genome editing tools particularly CRISPR/Cas9 have revolutionized plant genome manipulation for engineering resilience against changing climatic conditions, disease infestation, as well as functional genomic studies. CRISPR-mediated genome editing allows for editing at a single as well as multiple locations in the genome simultaneously, making it an effective tool for polyploid species too. However, still, its applications are limited to the model crops only. Extending it to crop plants will help improve field crops against the changing climates more rapidly and precisely. Here we describe the protocol for editing the genome of a field crop Brassica juncea (mustard), an allotetraploid and important oilseed crop of the Indo-Pak Subcontinent region. This protocol is based on the Agrobacterium-mediated transformation for the delivery of CRISPR components into the plant genome using cotyledon as explants. We elaborate on steps for recovering genome-edited knockouts, for validation of the edits, as well as recovering the transgene-free edited plants through a commonly used segregating approach.

Studying Salt-Induced Shifts in Gene Expression Patterns of Glucosinolate Transporters and Glucosinolate Accumulation in Two Contrasting Brassica Species.

Brassica crops are well known for the accumulation of glucosinolates-secondary metabolites crucial for plants' adaptation to various stresses. Glucosinolates also functioning as defence compounds pose challenges to food quality due to their goitrogenic properties. Their disruption leaves plants susceptible to insect pests and diseases. Hence, a targeted reduction in seed glucosinolate content is of paramount importance to increase food acceptance. GLUCOSINOLATE TRANSPORTERS (GTRs) present a promising avenue for selectively reducing glucosinolate concentrations in seeds while preserving biosynthesis elsewhere. In this study, 54 putative GTR protein sequences found in Brassica were retrieved, employing Arabidopsis GTR1 and GTR2 templates. Comprehensive bioinformatics analyses, encompassing gene structure organization, domain analysis, motif assessments, promoter analysis, and cis-regulatory elements, affirmed the existence of transporter domains and stress-related regulatory elements. Phylogenetic analysis revealed patterns of conservation and divergence across species. Glucosinolates have been shown to increase under stress conditions, indicating a potential role in stress response. To elucidate the role of GTRs in glucosinolate transportation under NaCl stress in two distinct Brassica species, B. juncea and B. napus, plants were subjected to 0, 100, or 200 mM NaCl. Based on the literature, key GTR genes were chosen and their expression across various plant parts was assessed. Both species displayed divergent trends in their biochemical profiles as well as glucosinolate contents under elevated salt stress conditions. Statistical modelling identified significant contributors to glucosinolate variations, guiding the development of targeted breeding strategies for low-glucosinolate varieties. Notably, GTR2A2 exhibited pronounced expressions in stems, contributing approximately 52% to glucosinolate content variance, while GTR2B1/C2 displayed significant expression in flowers. Additionally, GTR2A1 and GTR1A2/B1 demonstrated noteworthy expression in roots. This study enhances our understanding of glucosinolate regulation under stress conditions, offering avenues to improve Brassica crop quality and resilience.

Targeted genome editing in polyploids: lessons from Brassica.

CRISPR-mediated genome editing has emerged as a powerful tool for creating targeted mutations in the genome for various applications, including studying gene functions, engineering resilience against biotic and abiotic stresses, and increasing yield and quality. However, its utilization is limited to model crops for which well-annotated genome sequences are available. Many crops of dietary and economic importance, such as wheat, cotton, rapeseed-mustard, and potato, are polyploids with complex genomes. Therefore, progress in these crops has been hampered due to genome complexity. Excellent work has been conducted on some species of Brassica for its improvement through genome editing. Although excellent work has been conducted on some species of Brassica for genome improvement through editing, work on polyploid crops, including U's triangle species, holds numerous implications for improving other polyploid crops. In this review, we summarize key examples from genome editing work done on Brassica and discuss important considerations for deploying CRISPR-mediated genome editing more efficiently in other polyploid crops for improvement.

Salt-Induced Modulation of Ion Transport and PSII Photoprotection Determine the Salinity Tolerance of Amphidiploid Brassicas.

Brassica species show varying levels of resistance to salt stress. To understand the genetics underlying these differential stress tolerance patterns in Brassicas, we exposed two widely cultivated amphidiploid Brassica species having different genomes, Brassica juncea (AABB, n = 18) and Brassica napus (AACC, n = 19), to elevated levels of NaCl concentration (300 mM, half the salinity of seawater). B. juncea produced more biomass, an increased chlorophyll content, and fewer accumulated sodium (Na+) and chloride (Cl-) ions in its photosynthesizing tissues. Chlorophyll fluorescence assays revealed that the reaction centers of PSII of B. juncea were more photoprotected and hence more active than those of B. napus under NaCl stress, which, in turn, resulted in a better PSII quantum efficiency, better utilization of photochemical energy with significantly reduced energy loss, and higher electron transport rates, even under stressful conditions. The expression of key genes responsible for salt tolerance (NHX1 and AVP1, which are nuclear-encoded) and photosynthesis (psbA, psaA, petB, and rbcL, which are chloroplast-encoded) were monitored for their genetic differences underlying stress tolerance. Under NaCl stress, the expression of NHX1, D1, and Rubisco increased several folds in B. juncea plants compared to B. napus, highlighting differences in genetics between these two Brassicas. The higher photosynthetic potential under stress suggests that B. juncea is a promising candidate for genetic modifications and its cultivation on marginal lands.

Cliniciopathological Spectrum of Skin and Soft Tissue Swellings at a Tertiary Care Hospital of Lahore, Pakistan.

Background Skin and soft tissue swellings (SSTS) frequently present in dermatology, plastic surgery, and general surgery departments. While a general surgeon can take care of excisable lesions, people typically seek plastic surgery for cosmetic reasons. According to the signs and symptoms, soft tissue and skin lesions must be removed, and it is crucial to maintain cosmesis following their removal. Objective The aim of this article is to describe the clinical and histopathological types, sites, laterality, and postoperative complications of SSTS. Material and methods This retrospective study was conducted at the Department of Plastic and Reconstructive Surgery, Lahore General Hospital, Lahore, Pakistan in November 2022. We studied admitted patients from July 1, 2020 to June 30, 2022 for SSTS excision. Data on patients' demographics, associated features of SSTS, and their postoperative complications was gathered using Google Docs-generated proforma and sent to a statistician for the computation of results via a Microsoft Excel-generated spreadsheet. Results Out of the total 60 patients, 66.7% of the lesions were found in women. The mean age at presentation came out to be 34.16±17.42 years. Nevi with 16.7% were the most common SSTS in our study. The most common site of presentation of SSTS was the scalp and face in 63.3% of cases. Fever was the most frequently encountered post-excision complication in 40% of patients. Conclusion A comprehensive history, clinical examination, signs and symptoms, and the histology of the lesion, all play a crucial role in the management of such swellings. Surgery was the definitive treatment option for SSTS. There were very few major complications in a handful of patients.

Can The Anterolateral Thigh Flap Replace The Rectus Abdominis Free Flap In The Reconstruction Of Complex Maxillary Defects?

BACKGROUND: Maxilla is perhaps the most essential and visible part of the mid-face. It is a threedimensional structure and when reconstructing maxillectomy defects the principles of aesthetics as well as the best functional outcomes are taken into account. The aim of this study is to compare the Anterolateral Thigh Flap (ALTF) to the standard option like the Rectus Abdominis Free Flap (RAMFF) for the reconstruction of complex maxillary defects. METHODS: This descriptive case series was conducted at the Department of Plastic and Reconstructive Surgery, Shifa International Hospital Islamabad, Pakistan from 2009 to 2016. Patients of all age groups with complex maxillectomy defects, (Type III and IV according to Cordeiro classification) resulting from tumour resection, trauma, osteoradionecrosis or infection, underwent reconstruction with the free anterolateral thigh flap and the rectus abdominis free flap. RESULTS: Over a period of 8 years, 49 Rectus Abdominis free flaps and 32 Anterolateral thigh free flaps were performed for reconstruction of Type III and IV maxillectomy defects. The follow up was weekly for 1 month and then 3 monthly for the 1st year, 6 monthly for 2nd year and then yearly. All the patients had an uneventful immediate recovery. CONCLUSIONS: ALTF has advantages over the RAMFF in terms of the donor site morbidity, operative time and postoperative recovery in the reconstruction of complex maxillectomy defects.

Use Of Steroids In Rhinoplasty With Lateral Osteotomies For Reducing Post Operative Oedema.

BACKGROUND: TPostoperative periorbital oedema is a commonly encountered side effect of rhinoplasties in which lateral osteotomies have been incorporated. It dissatisfies the surgeon as well as the patient. Osteotomies are done at the end of all soft tissue manipulation to reduce the development of oedema. The aim of this study was to determine the efficacy of intravenous dexamethasone in reducing oedema in patients who undergo rhinoplasty with lateral osteotomies. METHODS: A Prospective randomized controlled trial was done at department of plastic and reconstructive surgery, Shifa International Hospital Islamabad. Sixty patients age between 16-55 requiring open rhinoplasty were taken for this study and divided in two groups. One group received dexamethasone 8mg intravenously preoperatively and second dose 4 hours postoperatively. The second group did not receive anything. Both groups were assessed on first post-operative day and 7th day for periorbital oedema. RESULTS: The overall decrease in oedema in patients who received steroid was by 50% while in control group was 33.3%. By the 7th day control group 13.3% patients had grade III oedema as compared to 3.33% in steroid group. Chi test was applied and p-value of 0.0289 was obtained which was found to be statistically very significant. CONCLUSIONS: Dexamethasone used in minimal dosage showed significant advantage in reducing periorbital oedema after rhinoplasty with no evidence of any side effects secondary to steroid administration.

Agreement between serology and histology for detection of Helicobacter pylori infection.

OBJECTIVE: To determine the percentage agreement between serology and histology for detection of Helicobacter (H.) pylori infection. STUDY DESIGN: Cross-sectional analytical study. PLACE AND DURATION OF STUDY: Department of Pathology and Microbiology, The Aga Khan University and Hospital, Karachi, from January to December 2009. METHODOLOGY: Fifty subjects were selected by non-probability purposive sampling from laboratory data who had serological testing of H. pylori IgG antibody, prior to histological evaluation of endoscopic gastric or/and duodenal biopsies. Serological Quantification of H. pylori IgG was carried out with HpG screen ELISA kit (Genesis Diagnostics, UK), using an enzyme linked immunosorbent assay for detection of IgG antibodies against H. pylori. Manufacturer's recommended cutoff value was used and results were considered positive when greater than 7 U/ml. For histological diagnosis, an expert histopathologist characterized the presence of spiral bacteria in the mucosal layer or the surface of epithelial cells on microscopic examination, as a positive test. RESULTS: An agreement of 0.72 was found by Kappa statistics between serology and histopathology results and a good diagnostic accuracy (86%) of serological testing was observed for the diagnosis of H. pylori infection. CONCLUSION: A substantial agreement was found between serology and histopathology results to detect the H. pylori infection. Laboratory-based serologic testing using ELISA technology to detect IgG antibodies is inexpensive, noninvasive and convenient method to detect the H. pylori infection in primary care setting.

Gastrointestinal, liver and biliary tract pathology: a histopathological and epidemiological perspective from Pakistan with a review of the literature.

AIM: To present an epidemiological and histological perspective of diseases of the gastrointestinal tract (including liver and biliary tract) at the Section of Histopathology, Department of Pathology, AKUH, Karachi, Pakistan. MATERIALS AND METHODS: All consecutive endoscopic biopsies and resections between October 1 and December 31, 2012 were included. RESULTS: A total of 2,323 cases were included. Carcinoma was overwhelmingly the commonest diagnosis on esophageal biopsies (69.1%); chronic helicobacter gastritis (45.6%) followed by adenocarcinoma (23.5%) were the commonest diagnoses on gastric biopsies; adenocarcinoma (27.3%) followed by ulcerative colitis (13.1%) were the commonest diagnoses on colonic biopsies; acute appendicitis (59.1%) was the commonest diagnosis on appendicectomy specimens; chronic viral hepatitis (44.8%) followed by hepatocellular carcinoma (23.4%) were the commonest diagnoses on liver biopsies; chronic cholecystitis was the commonest diagnosis (over 89%) on cholecystectomy specimens. CONCLUSIONS: Squamous cell carcinoma comprised 88.8% of esophageal cancers. About 67% were in the lower third and 56.5% were moderately differentiated; mean ages 49.8 years for females and 55.8 years for males; 66% cases were from South West Pakistan. Over 67% patients with gastric adenocarcinoma were males; mean ages 59 and 44 years in males and females respectively, about 74% gastric carcinomas were poorly differentiated; and 62.2% were located in the antropyloric region. About 63% patients with colorectal adenocarcinoma were males; mean ages 46.1 and 50.5 years for males and females respectively; tumor grade was moderately differentiated in 54%; over 80% were located in the left colon. In 21.2% appendicectomies, no acute inflammation was found. Acute appendicitis was most common in young people. Hepatitis C (66.3%) was more common than hepatitis B (33.7%); about 78% cases of hepatocellular carcinoma occurred in males; females comprised 76.7% patients with chronic cholecystitis; and 77.8% patients with gall bladder carcinoma. All resection specimens showed advanced cancers. Most cancers occurred after the age of 50 years.

Hodgkin lymphoma in Pakistan: an analysis of subtypes and their correlation with Epstein Barr virus.

The epidemiology of Hodgkin lymphoma (HL) shows a wide geographic variation with regard to age, gender, histological subtypes and their association with Epstein-Barr virus. The proportion of EBV positive cases appears higher in developing than in developed countries. EBV is a common infection in Pakistan due to poor socioeconomic conditions, but reports regarding HL subtypes have been rather selective. Our aims were to establish the relative frequencies of the five subtypes of Hodgkin lymphoma, to determine their associations with Epstein-Barr virus, and finally to establish whether such association follows patterns seen in developing or developed countries. Among 100 cases, the male: female ratio was 4.5:1, with an age range of 4-82 years and an average of 26.6 years. Similar to the subtype distribution in developing countries, mixed cellularity was the commonest 57%, followed by nodular sclerosis 35%, lymphocyte rich 6% and nodular lymphocyte predominant 2%. EBV-LMP1 staining was demonstrated in 41/57 (71%) of the mixed cellularity and the 19/35 (54.2%) of nodular sclerosis subtypes. All 6 cases of lymphocyte rich and 2 cases of nodular lymphocyte predominant were negative for EBV-LMP 1. Speculation about prognostic effects of EBV infection on the course of HL are tempting. Thus the EBV-positive HL could in the future prove to be an excellent candidate for targeted cellular immunotherapy.

PEComa in a 12-year-old boy.

PEComas (perivascular epithelioid cell tumours) are rare mesenchymal tumours, characterised by epithelioid, clear cell morphology with coexpression of melanocytic and smooth muscle markers. They are usually seen in adult females in a variety of anatomical locations, of which, kidney, lung and the gynaecologic tract are the most common. We present a case of PEComa arising from the kidney of a 12-year-old boy.

Iron, folate and cobalamin deficiency in anaemic pregnant females in tertiary care centre at Rawalpindi.

BACKGROUND: Anaemia in pregnancy is a common clinical problem contributing to increased maternal and foetal morbidity. This study was carried out to determine frequency of iron, folate and cobalamin deficiency and associated risk factors in the anaemic pregnant females who reported first time during second and third trimester for antenatal check-up in the tertiary care hospital at Rawalpindi. METHODS: This case control study was carried out in a tertiary care hospital at Rawalpindi. Two hundred and fifty pregnant women (age: 19-43 years) consisting of 125 anaemic (Hb < 110 g/L) and 125 non-anaemic who reported first time at antenatal clinic were included. Data on socio-demographic characteristics, parity and dietary intake were collected. Complete blood counts were done. Serum ferritin, folate and cobalamin assays were performed by using DPC kits on Immulite-1000. RESULTS: The pregnant women were categorised having mild (Hb up to 54%), moderate (Hb up to 36%), or severe (Hb up to 10%) anaemia during antennal visit. They had significantly lower median (range) levels of haemoglobin 96 (40-110) g/L, ferritin 8 (3-142) microg/L, folate 15 (3-54) etamol/L and cobalamin 171 (111-629) etamolL than controls (p = < 0.01). Micronutrient analysis revealed secondary pregnancy related deficiency of Iron (57%), folate (20%). combined iron and folate (19%) and cobalamin (4%) in the female Among the risk factors, low income (OR: 7.69), multiparty (OR: 2.93), lack of iron/folate supplementation (OR 2.91) and inadequate dietary intakes (OR 2.51) were associated with anaemia. CONCLUSION: The pregnant anaemic women had iron (57%); folate (20%), followed by combined iron folate (19%) and cobalamin (4%) deficiency during first antenatal visit. Low income, multiparty, poor diet and lack of supplements are the main contributor in development of anaemia during pregnancy.

Minimally invasive papillary carcinoma of the extrahepatic bile ducts.

Invasive papillary carcinomas of the Extrahepatic Bile Ducts (EBD) are uncommon (4-5%). The course is less aggressive than conventional adenocarcinomas of the extrahepatic bile ducts. Non-invasive and minimally invasive papillary carcinomas are even rarer, behave as in-situ carcinomas and are associated with excellent long-term prognosis. A variety of lesions of the EBD that show papillary architecture should be distinguished from papillary carcinoma. Here, we report a case of papillary carcinoma of the common bile duct showing minimal invasion. Separation of invasive from non-invasive or minimally invasive papillary carcinoma is critical in estimating the patient outcome.