Machine-learning-optimized Cas12a barcoding enables the recovery of single-cell lineages and transcriptional profiles.

The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes-at a fraction of the size of Cas9 barcodes-achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.

Bone allograft block using demineralized freeze-dried bone ring technique in Indian patients.

Our study aimed to evaluate the effectiveness of the bone ring technique for ridge augmentation using Demineralized Freeze - Dried Bone Allograft block in Siebert's class II/class III defects along with simultaneous implant placement. A total of 15 partially edentulous patients (16 surgical sites) with Siebert's class II and/or class III defects in the anterior region of both jaws requiring ridge augmentation along with implant placement were selected. Starting from the first stage, surgery (Ridge augmentation+ implant placement) was done in the first month. Then, with continuous follow-ups and radiographic assessment, after 6 months of 2nd stage surgery was done, the implant was loaded with the final restoration. Significant results were revealed with all the parameters other than keratinized gingival and peri-implant mucosa thickness. With the mean bone resorption of 1.22 mm and 1.17 mm at the mesial and distal site at a 6-month interval, the success rate of the bone ring technique was 93.75%. The allograft bone ring technique showed a favorable outcome for the reconstruction of large vertical defects.

Dermoscopy in Leprosy: A Clinical and Histopathological Correlation Study.

BACKGROUND: Leprosy, an insidious infectious granulomatous disease, is diagnosed traditionally through clinical examination coupled with skin smears and histopathology. It has myriad clinical presentations that pose diagnostic challenges. Lately, dermoscopy has emerged as a rapid, noninvasive diagnostic modality for many dermatoses. OBJECTIVES: We evaluated the dermoscopic findings of various manifestations of leprosy and correlated them with clinical and histopathological features. METHODS: This prospective, cross-sectional study was conducted in our skin outpatient department for a period of 1 year. Patients newly diagnosed as having leprosy or those undergoing leprosy treatment for less than 6 months were included. The most representative lesion was dermoscopically evaluated and later biopsied. RESULTS: We included 73 patients in the study. Results indicated an obvious correlation between dermoscopic findings and histopathology. We noted orangish yellow and white structureless areas, steadily throughout the spectrum, depicting dermal granuloma. Additionally, we observed focal vascular structures such as branching, linear, and crown vessels that result from the pressure of granuloma pushing the dilated vessels upwards. The relative absence of skin appendages aided in differentiating leprosy from other granulomatous disorders. Novel findings of our study were the detection of a branch-like pattern of clofazimine-induced pigmentation on dermoscopy and orange globules on onychoscopy. Other unique findings included violaceous structureless areas, characteristic large telangiectatic vessels, follicular plugging, star-shaped silvery-white scaling, and white globules in type 1 reaction; white shiny steaks were observed in patients with borderline lepromatous leprosy, and central white dots and keratotic plugs were observed in patients with histoid leprosy. CONCLUSIONS: Dermoscopy, as a noninvasive modality, could aid in the quick diagnosis of leprosy and should be used as a handy tool to complement other investigative tools for this disease.

Endocrinological Testicular Dysfunction in Patients with Lepromatous Leprosy and the Impact of Disease on Patient's Quality of Life.

INTRODUCTION: Leprosy or Hansen's disease poses a drastic impact on the quality of life in affected patients even after successful completion of treatment. The involvement of the endocrine system in leprosy is usually insidious, silent, and under-reported, especially the testicular dysfunction. AIMS AND OBJECTIVES: The present study was aimed at evaluating the abnormalities of the primary testicular hormone testosterone and the gonadotrophins LH and FSH in male patients affected with lepromatous leprosy and assessing the impact of the disease on quality of life (QOL). MATERIALS AND METHODS: The study included 43 married male patients diagnosed with lepromatous leprosy. Careful history taking and examination for symptoms of testicular dysfunction were done. Serum concentrations of total testosterone, FSH, and LH were noted. The QOL was evaluated using the WHO Quality of Life-BREF (WHOQoL-BREF). RESULTS: The most common clinical manifestation of testicular dysfunction was reduced or loss of libido reported in 12 (27.9%) patients followed by gynaecomastia in 7 (16.3%). Ultrasonographic (USG) analysis revealed reduced testicular volume in 31 (72.1%) patients, and average testicular volume was 11.9 ± 4.9 mL each. Seventeen (39.5%) patients had low serum testosterone levels, 9 (20.9%) had high serum FSH level, and 11 (25.6%) high LH levels. There was a significant negative correlation between testosterone level and FSH as well as LH. There was also a significantly positive correlation between testicular volume and testosterone level. Symptomatic patients with gynaecomastia/gynaecothelia had higher hormonal derangement than those who had other symptoms. On QOL, most patients scored lowest on the domain of "social relationships" (including sexual wellbeing) followed by "psychological health". CONCLUSION: We found a high rate of USG diagnosed testicular atrophy in lepromatous leprosy patients. Therefore, every leprosy patient should be thoroughly examined clinically to rule out features of testicular dysfunction. Testicular function tests should be routinely carried out in all leprosy patients to arrive at an early diagnosis. Leprosy is found to affect all domains of a patient's quality of life.

A Comparative Study between Follicular Unit Transplantation and Autologous Non-cultured Non-trypsinized Epidermal Cells Grafting (Jodhpur Technique) in Stable Vitiligo.

INTRODUCTION: Several modalities are currently available for the treatment of vitiligo, but generally they do not result in complete cure of the disease. Despite the limitations and some side effects, surgical modalities appear to be the method of choice in recalcitrant stable vitiligo. Both Jodhpur technique (JT) and follicular unit transplantation (FUT) are simpler and cheaper methods, requiring minimal infrastructure. MATERIALS AND METHODS: Hundred patches in 30 patients were divided into Group A and Group B, that is, hair follicle transplantation and autologous non-cultured non-trypsinized epidermal cell suspension grafting (JT), respectively. Fifty patches were included in each group. Efficacy of both the techniques, extent of pigmentation, and color match assessment were the main objectives. RESULTS: Excellent repigmentation (>75%), at the end of 20 weeks post surgery, was observed in 70% lesions in FUT group and 72% of lesions in JT group. Good response (extent of repigmentation of 50%-75%) was seen in 18% of lesions in FUT group as compared to 26% in JT group. JT and FUT technique were almost equal in producing excellent repigmentation. Color match was same for both the groups, that is, 49 of 50 patches showed same color as surrounding in both groups at the end of 20 weeks. Repigmentation of the depigmented hairs occurred in 11 of 46 patients with associated leukotrichia. CONCLUSION: Our study indicates that both JT and FUT are safe and effective techniques in terms of repigmentation but JT is somewhat superior to FUT in producing excellent and good repigmentation, side effects, patient satisfaction, and dermatology life quality index reduction, while the color match was almost same with both techniques.

Anterior Ectopic Cilia with Myopia and Lichen Nitidus: A Rare Case Report with Dermoscopic Findings.

Ectopic cilia, or lash follicles situated over abnormal sites, are an extremely rare entity. Here, we report the case of a 6-year-old-boy who presented with ectopic cilia over the left upper eyelid along with a positive family history. Dermoscopy revealed discreet terminal hair emerging from the individual hair follicles surmounted over a diffuse structureless whitish-yellow zone and a few vellus hairs in the surrounding normal skin. These hairs, on histopathological examination, had multiple pilosebaceous follicular units embedded in a desmoplastic stroma with multiple eccrine sweat glands. Other incidental findings included lichen nitidus and myopia.

Changes and Compromises in Health Choices during COVID-19 Lockdown in Kathmandu Valley: A Descriptive Cross-sectional Study.

INTRODUCTION: Nepal government enforced a lockdown as a social distancing measure to curb the COVID-19 pandemic. The lockdown has led to compromises in day to day choices like food, exercise, sleep, self-care routines and utilization of healthcare facilities - directly and indirectly influencing aspects of health. Identification of compromised health choices can assist in better planning of inevitable future crises. METHODS: This is a cross-sectional descriptive study based on an online self -administered questionnaire, done using CHERRIES criteria, conducted from March 30, 2020, to July 31, 2020. Ethical approval for the study was obtained from the Institutional Review Committee of Nepal Health Research Council (Registration number: 2119; 300/2020 P). Descriptive statistics was used for analysis. RESULTS: Our study had 340 (51%) female and 325 (48.7%) male participants. A total of 112 (67.9%) reported decreased consumption of tobacco and 178 (53.6%) reported decreased consumption of alcohol during the lockdown period. Participants who reported that they would have visited a hospital if they had a flu-like illness increased from 151 (22.6%) pre-pandemic to 391 (58.6%) post-pandemic. Increase in news consumption was reported by 528 (79.2%). Out of 43 (6.4%) participants with a chronic condition, 30 (69.8%) reported having missed follow up due to the lockdown. CONCLUSIONS: The health of an individual is determined by various choices s/he makes on a day to day basis. Many of those choices are in turn influenced by the availability and accessibility of commodities. Lessons learned from the affected lives due to COVID-19 can be used in proper planning of inevitable future crises.

Broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics.

Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To define bNAb targets, we characterized 28 antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified J9 and J8, two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies showed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against DENV with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested that J9 and J8 followed divergent somatic hypermutation pathways, and that a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a new epitope that can be exploited for vaccine design.

Preliminary Study on Serum Lactate Dehydrogenase (LDH)-Prognostic Biomarker in Carcinoma Breast.

INTRODUCTION: Serum Lactate Dehydrogenase (LDH) is one of the biochemical markers for breast cancer. Serum LDH is enzyme required for anaerobic glycolysis. One of its isoenzyme is increased in breast cancer due to up-regulation in its gene. It leads to increase in serum LDH level in breast cancer patients. Serum LDH is economical, easily available and easy to estimate. AIM: In the present study, we evaluated the LDH levels in circulation of newly diagnosed patients of breast cancer and tried to correlate it with different TNM staging of carcinoma breast before interventions and after adjuvant therapy of these patients. MATERIALS AND METHODS: This prospective study was done on 83 diagnosed patients of breast cancer was conducted among poor patients in rural area. This study was conducted in the Department of Surgery between October 2008 to October 2010, at MGIMS, Sevagram, Maharashtra, a rural medical college located in Central India. Out of total 83 participants, 10 participants were having adverse events following surgery and remaining 73 participants were without adverse events following surgery. The significant difference in serum LDH levels between two groups, with and without adverse surgical outcome was calculated by Mann-Whitney U test. RESULTS: Patients with higher clinical TNM staging were having higher serum LDH levels. The serum LDH levels at sixth months following surgery showed a trend of statistically significant difference between patients with and without adverse events. As increased serum LDH levels in breast cancer patients shows poor prognosis, surgical outcome or advanced metastases. CONCLUSION: Serum LDH monitoring can be used as a prognostic biomarker in patients of breast cancer. For confirmation of this finding, we require further more studies on larger sample size and long-term follow-up in patients specifically with higher serum LDH levels.

Persistent Müllerian Duct Syndrome (PMDS): a Rare Anomaly the General Surgeon Must Know About.

Persistent Müllerian duct syndrome is a rare condition occasionally encountered in men with normal phenotype but with presence of Müllerian duct structures. In India, owing to neglect and lack of facilities, we encounter this condition in adult males. We encountered on the same day in the operation theatre two phenotypic males aged 40 years and 10 months who had inguinal hernia on one side along with contralateral undescended testis. Both patients intraoperatively had uterus with fallopian tubes and underwent subtotal hysterectomy with preservation of vas. Repair of inguinal hernia with fixation of the testis in the scrotum was done. Though rare, every surgeon operating upon inguinal hernia or undescended testes or cryptorchidism needs to know about the presence of the uterus in a phenotypic male patient at any age. High degree of suspicion and awareness is needed to diagnose this condition. Early treatment is needed to maintain fertility and to prevent the occurrence of malignancy in remnant müllerian structures.

Histopathological evaluation of collagen fibers using picrosirius red stain and polarizing microscopy in oral squamous cell carcinoma.

BACKGROUND: Collagen is a part of structural connective tissue in every organ of the body. Study of collagen brings into focus the current knowledge of biosynthesis and degradation as a complex mechanism. One of the major aspects of tumor cell invasion and metastasis is the interaction between cancer cells and extracellular matrix component. Property of picrosirius red to enhance the birefringence is the result of basic protein nature of collagen molecules. There exists a direct relationship between the presence of type IV collagen and degree of differentiation in squamous cell carcinoma (SCC) cells as they lose their capability to form the basement membrane. MATERIALS AND METHODS: Thirty cases of oral SCC (OSCC) were evaluated for standard staining protocol of picrosirius red to analyze collagen fibers in different grades of OSCC under polarizing microscopy. RESULTS: In this study, moderately to poorly differentiated OSCC cases showed a gradual change in polarizing colors from yellowish orange to greenish yellow particularly in the vicinity of invading tumor islands. Thick collagen fibers forming bundles in parallel disposition were found around the neoplastic areas in discontinuous fashion. Type I collagen fibers of different lengths were strongly birefringent and swirl pattern, parallel orientation was evident. CONCLUSION: An observable stromal change with the progression of neoplasm was evinced with picrosirius red stain in different thickness of collagens with a significant change in the arrangement from the early stage to the advanced stage according to tumorogenesis.

Transgenic plants over-expressing insect-specific microRNA acquire insecticidal activity against Helicoverpa armigera: an alternative to Bt-toxin technology.

The success of Bt transgenics in controlling predation of crops has been tempered by sporadic emergence of resistance in targeted insect larvae. Such emerging threats have prompted the search for novel insecticidal molecules that are specific and could be expressed through plants. We have resorted to small RNA-based technology for an investigative search and focused our attention to an insect-specific miRNA that interferes with the insect molting process resulting in the death of the larvae. In this study, we report the designing of a vector that produces artificial microRNA (amiR), namely amiR-24, which targets the chitinase gene of Helicoverpa armigera. This vector was used as transgene in tobacco. Northern blot and real-time analysis revealed the high level expression of amiR-24 in transgenic tobacco plants. Larvae feeding on the transgenic plants ceased to molt further and eventually died. Our results demonstrate that transgenic tobacco plants can express amiR-24 insectice specific to H. armigera.

Role of Laparoscopy in Chronic and Recurrent Abdominal Pain-Rural Area Experience.

Chronic and recurrent abdominal pain is a common problem faced by the medical specialist. It leads to physical and psychological disability in a person. Despite radiological and clinical investigations when diagnosis cannot be ascertained then laparoscopy is one of the modalities that could be of benefit. We aim to evaluate the diagnostic and therapeutic value of laparoscopy in cases with chronic abdominal pain. Sixty-seven patients who attended the outpatient department of MGIMS, Sewagram with chronic and recurrent abdominal pain were included in this prospective study. All patients underwent preoperative workup and were subjected to laparoscopic evaluation. The commonest site of pain was the periumbilical region and nausea was the most common accompanying clinical feature. Adhesions were the most common laparoscopic findings (48.3 %) followed by appendiceal pathology (14.6 %). Abdominal Kochs which is predominant in developing countries was seen in 11.4 % of the cases. Laparoscopy is an effective diagnostic and therapeutic modality in the management of patients with chronic abdominal pain.