Clinico-pathologic profile and outcomes of pediatric endocrine patients managed by endocrine surgeons: Experience over three decades in a tertiary center in India.

BACKGROUND: Pediatric endocrine disorders requiring surgical intervention are rare and so are experienced surgeons dealing with these. The aim of the current study was to investigate disease profile and perioperative outcome of pediatric patients with surgical endocrine disorders in an endocrine surgery unit. METHODS: This retrospective study (Sep 1989-Aug 2019) consisted of pediatric endocrine surgery patients (<18 years) who were managed by a team of pediatric endocrinologists and endocrine surgeons at our center. Patients were divided into three cohorts consisting of a decade each. Clinico-pathologic variables, perioperative events operative and follow-up details were recorded. RESULTS: A total of 332 children were included and their mean age was 14.6 ± 3.9 years (M:F = 1:1.6). Thyroid disorders were most prevalent (59.8%), followed by adrenal (28.2%), parathyroid (10.4%), and pancreas (1.5%). Incidence of benign, malignant, and congenital/developmental disorders were 65.4, 28.1 and 8.3, respectively. Familial association was observed in 8.9% children, which is highest among pheochromocytoma patients. Overall, 201 thyroidectomies + associated procedures, 35 parathyroidectomies, 96 adrenal and paraganglioma resections, and 5 pancreatic procedures were performed. Median hospital stay was 5.6 ± 4.1 days. The number of cases increased significantly over 3 decades. Clinical profile and outcome did not vary except for significant decrease in incidence of malignant pathology (p = 0.04) and increase in VHL cases (p = 0.04) in the last decade though overall increase in familial cases was nonsignificant (p = 0.11). No perioperative mortality was observed except for 3% after adrenalectomy. CONCLUSION: A team of dedicated endocrine surgeons and pediatric endocrinologists is effective in management of pediatric endocrine surgery.

Optimization of dilution rate and mixed carbon feed for continuous production of recombinant plant sucrose:sucrose 1-fructosyltransferase in Komagataella phaffii.

The trisaccharide 1-kestose, a major constituent of commercial fructooligosaccharide (FOS) formulations, shows a superior prebiotic effect compared to higher-chain FOS. The plant sucrose:sucrose 1-fructosyltransferases (1-SST) are extensively used for selective synthesis of lower chain FOS. In this study, enhanced recombinant (r) 1-SST production was achieved in Komagataella phaffii (formerly Pichia pastoris) containing three copies of a codon-optimized Festuca arundinacea 1-SST gene. R1-SST production reached 47 U/mL at the shake-flask level after a 96-h methanol induction phase. A chemostat-based strain characterization methodology was adopted to assess the influence of specific growth rate (µ) on cell-specific r1-SST productivity (Qp) and cell-specific oxygen uptake rate (Qo) under two different feeding strategies across dilution rates from 0.02 to 0.05 h-1. The methanol-sorbitol co-feeding strategy significantly reduced Qo by 46 ± 2.4% compared to methanol-only feeding without compromising r1-SST productivity. Based on the data, a dilution rate of 0.025 h-1 was applied for continuous cultivation of recombinant cells to achieve a sustained r1-SST productivity of 5000 ± 64.4 U/L/h for 15 days.

Cardiac Changes and Their Reversal Following Curative Surgery in Pheochromocytoma: PheoCard Prospective Cohort Study.

BACKGROUND: Pheochromocytoma and paraganglioma (PPGL) are catecholamine producing tumors of chromaffin cell origin, known to cause varied cardiovascular manifestations from hypertension to myocardial infarction. This study sought to objectively evaluate the cardiac changes in PPGL patients and their reversal following curative surgery. METHODS: The PheoCard study was registered in ClinicalTrials.gov (NCT05082311) and involved 35 consecutive PPGL patients managed as per standard protocol involving alpha blockade followed by curative surgery. They underwent detailed cardiac evaluation using 2D-echocardiography and speckle tracking echocardiography at the time of diagnosis, 7-10 days after alpha blockade, and at 7 days, 3 months, and 6 months after surgical removal. Age- and gender-matched essential hypertensives and healthy individuals (10 in each group) served as two control groups. RESULTS: Patients with PPGLs had significantly higher mean blood pressure, left ventricle end-diastolic dimension and volume (LVEDD, LVEDV), left ventricle end-systolic volume (LVESV), septal wall thickness, LV hypertrophy, lower mean LV ejection fraction (LVEF), early diastolic mitral annular velocity (E/A), decreased amplitude of LV longitudinal strain, and increased circumferential strain (p < 0.001) when compared with the control groups at baseline. After alpha blockade, there was marked reduction in the mean LVEDD, LVEDV, LVESV, and normalization of E/A ratio (p < 0.001) in the PPGL patients. Following curative surgery (normalization of fractionated urinary metanephrines at 7-10 days post-operatively), there was early improvement in all echocardiographic parameters and it continued to improve even at 6 months after surgery. There was marked improvement in the global longitudinal strain as seen on serial speckle tracking echocardiography with recovery of most of the segments of LV depicting the reversal of subclinical endocardial dysfunction (p < 0.001). CONCLUSION: PPGL patients despite normal systolic function have subclinical LV diastolic dysfunction which is reversed after curative surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT05082311.

Total vs less than total thyroidectomy for benign multinodular non-toxic goiter: an updated systematic review and meta-analysis.

BACKGROUND: We have performed an updated meta-analysis of randomized controlled trials (RCT) comparing total thyroidectomy (TT) with less than total thyroidectomy (LTT) for benign multinodular non-toxic goiter (BMNG). OBJECTIVES: The objective was to evaluate the effects and outcomes of TT as compared to LTT. METHODS: Eligibility criteria: RCTs comparing TT vs LTT. INFORMATION SOURCES: PubMed, Embase, Cochrane Library and online registers were searched for articles comparing TT with LTT. Risk of bias: Articles were assessed for risk of bias using the Cochrane's revised tool to assess risk of bias in randomized trials (RoB 2 tool). SYNTHESIS OF RESULTS: The main summary measures were risk difference using a random effects model. RESULTS: Five randomized controlled trials were included in the meta-analysis. Recurrence rate was lower for TT compared to LTT. Adverse events like temporary or permanent recurrent laryngeal nerve (RLN) palsy and permanent hypoparathyroidism were similar in both groups except for the rate of temporary hypoparathyroidism which was lower in the LTT group. DISCUSSION: All studies had unclear risk of bias for blinding of the participants and personnel and high risk of bias for certain selective reporting. This meta-analysis did not show any clear benefit or harm of either procedure (TT vs LTT) for goiter recurrence and re-operation rates (for both recurrence and incidental thyroid cancer). However, re-operation for goiter recurrence was significantly higher in the LTT group based on a single RCT. Evidence suggests increased rates of temporary hypoparathyroidism with TT but there was no difference in the rate of RLN palsy and permanent hypoparathyroidism between the two methods. The overall quality of evidence was low to moderate.

Production of high-value oxidative enzymes by Cyathus bulleri on agricultural and agri-food wastes for application in the textile sector.

Ligninolytic and other oxidative enzymes have emerged as promising biocatalysts in several industries. Since their production at a low cost is necessary for any large-scale application, we demonstrate the use of rice bran (RB), an agricultural waste and agri-food wastes such as potato peelings (PP), banana peelings (BP), and green pea peelings (GPP) for their production. High activity of laccase (12 U/ml), manganese peroxidase (16.11 ± 1.43 U/ml), and aryl alcohol oxidase (1.25 U/ml) was obtained on the PP on the 12th day of growth and ~ 6 U/ml of lytic polysaccharide monooxygenase was obtained on the 14th day of growth demonstrating PP to be a good substrate for their production. RB served as the next best substrate for the production of these enzymes. While the GPP was effective for the production of laccase (9.2 U/ml), this and the BP were not good substrates for the production of other enzymes. Efficient (48-82%) decolorization of several azo-, triarylmethane- dyes, and real textile effluent, without the addition of any mediator, demonstrated the high oxidative ability of the crude culture filtrate produced on the PP (CF-PP), which was a significant improvement compared to the treatment given by the previously reported culture filtrate obtained on wheat bran (CF-WB). An extensive breakdown of Reactive Orange (RO) 16 was demonstrated using CF-PP resulting in the formation of a new product at m/z of 294.05 (6-acetamido-3,4-dioxo-3,4-dihydronapthalene-2-sulfonate), previously reported to be produced on ozonation/advanced oxidation of RO16. The predominant laccase and manganese peroxidase isoforms produced on the PP were also identified.

Clinical, Biochemical, Tumoural and Mutation Profile of VHL- and MEN2A-Associated Pheochromocytoma: A Comparative Study.

OBJECTIVE: To compare clinical, biochemical, tumoural and mutational characteristics of Von Hippel Lindau Syndrome (VHL)-associated pheochromocytoma (PCC) to multiple endocrine neoplasia 2A (MEN2A)-associated pheochromocytoma. DESIGN: Retrospective study design in a tertiary health care centre in Northern India. METHODS: A total of 47 patients with biochemical and histologically proven pheochromocytoma/paraganglioma (PCC/PGL): 29 associated with VHL and 18 with MEN2A, were divided in two cohorts, respectively. Analysis of their medical records along with a prospective follow-up was done. RESULTS: There were more children <19 years in VHL group (13 vs 1). Despite majority of VHL-PCC showing elevation of normetanephrine (NMN) (93%) as compared to MEN2A-PCC (22.2%), 75.8% presented with hypertension as compared to MEN2A (33.3%). The average size of VHL-PCC tumours was 5.66 cm. VHL-PCC as compared to MEN2A-PCC were multifocal (75% vs 61.1%), bilateral synchronous (72.4 vs 61.1%) and extra-adrenal (17.2% vs 0%). Both VHL (24%) and MEN2A-PCC (27.7%) showed multiple nodules, but more MEN2A PCC showed extra-tumoural hyperplasia (44.4% vs. 6.8%). In VHL, the commonest mutation (n = 17) was missense mutation with a hot spot on exon 3, while in MEN2A-PCC majority (66.6%) had 634 mutation in exon 11 and only 2 patients had the rare 611 mutation in exon 10. CONCLUSION: In contrast to world literature, our study suggests Indian VHL-PCC can be symptomatic in spite of noradrenergic phenotype, large in size and multifocal. Multiple nodules in VHL-PCC could increase risk of recurrence following subtotal adrenalectomy.

Yeast-Mycelial Dimorphism in Pichia pastoris SMD1168 Is Triggered by Nutritional and Environmental Factors.

This study reports, for the first time, morphological transition from yeast-like to filamentous form, normally associated with pathogenicity/increased protein secretion, in Pichia pastoris SMD1168 strain. The response was recorded in response to nutritional and environmental cues. The factors affecting this switch were extracellular pH (under nitrogen starvation conditions), carbon and nitrogen source under nitrogen- and carbon-limiting conditions respectively. Under nitrogen-limiting conditions, addition of fructose and sucrose in the culture medium induced filamentous morphology in a segregated form whereas addition of galactose led to a mixture of yeast and the filamentous form of the cells. Under carbon-limiting conditions, isoleucine and proline forced a filamentous form whereas glycine, valine, alanine and phenylalanine promoted yeast-like morphology. Similar dimorphic shift was also displayed by a recombinant methanol slow utilizing (Muts) strain (SMD-GCSF Muts) producing human granulocyte colony-stimulating factor in response to change in the initial inoculum level. Analysis of the extracellular metabolome by GC-MS indicated that several amino acids (leucine, proline, tyrosine), carboxylic acids (phenylacetic-, propanoic acid), alcohols and butylamine were present at different levels in the culture broth of the two morphological forms. High accumulation of proline and butylamine was seen in the extracellular culture filtrate of the filamentous form of the yeast. Presence of quorum-sensing molecules (phenylethyl alcohol, dodecanol) suggested complex network of pathways involved in this morphological transition.

High level production of stable human serum albumin in Pichia pastoris and characterization of the recombinant product.

Human serum albumin (HSA) is an important therapeutic used in clinical settings for restoration of blood volume and treatment of chemotherapy induced neutropenia. Currently sourced from human serum, it carries the risk of contamination with viruses. The production of stable extracellular recombinant (r)HSA was achieved at nearly 1 g/L at shake-flask level in Pichia pastoris (syn. Komagataella phaffii) containing a three-copy containing HSA expression cassette, prepared in vitro. The HSA specific transcripts were increased by 1.82- to 2.46-fold in the three-copy containing clones indicating increased transcript levels to result in enhanced production of extracellular rHSA. The purified rHSA displayed secondary structure, zeta potential, size distribution and biological efficacy that matched with that of the commercial HSA. Cultivation strategy was developed at bioreactor level for the single HSA expression cassette containing recombinant which led to productivity of 300 mg/L/d of rHSA with minimum proteolytic cleavage.

An insight into transcriptome of Cyathus bulleri for lignocellulase expression on wheat bran.

To identify enzymes that can be effectively used for hydrolysis of lignocellulosic biomass, an attractive carbon source in biorefineries, transcriptome analysis was carried out of wheat bran grown fungus, Cyathus bulleri. A comprehensive set of transcripts, encoding carbohydrate active enzymes, were identified. These belonged to 55, 32, 12, 11 and 7 different families of the enzyme classes of Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Auxiliary Activities (AAs), Carbohydrate Esterases (CEs) and Polysaccharide Lyases (PLs) respectively. Higher levels of transcripts were obtained for proteins encoding cellulose and hemicellulose degrading activities (of the GH class) with the highest diversity found in the transcripts encoding the hemicellulases. Several transcripts encoding pectin degrading activity were also identified indicating close association of the pectin with the cellulose/hemicellulose in the cell wall of this fungus. Transcripts encoding ligninases were categorized into Cu radical oxidase, Glucose-Methanol-Choline oxidoreductase (with 37 different transcripts in the AA3 sub-family), Laccase and Manganese peroxidases. Temporal gene expression profile for laccase isoforms was studied to understand their role in lignin degradation. To our knowledge, this is the first analysis of the transcriptome of a member belonging to the family Nidulariaceae.

Pediatric Primary Hyperparathyroidism: Experience in a Tertiary Care Referral Center in a Developing Country Over Three Decades.

BACKGROUND: There is limited experience in managing pediatric primary hyperparathyroidism (PHPT). The aim of this study was to analyze the clinical presentation and outcome of surgery in children with PHPT managed at a tertiary referral center. METHODS: This retrospective study (September 1989-August 2019) consisted of 35 pediatric PHPT patients (< 18 years) who underwent parathyroidectomy. Clinico-pathologic profile and outcome were noted. RESULTS: The mean age of cohort was 15.2±2.9 years and girls outnumbered boys (M:F = 1:1.9). Familial and symptomatic disease was noted in 8.5 and 94.3% cases, respectively. Skeletal manifestations (83%) were the commonest followed by renal (29%). Fifty-four percent children had skeletal fractures, and 23% were bed-ridden. Among rare manifestations, hypercalcemic crisis, recurrent pancreatitis and stigmata of rickets were observed in 2.8, 11.4 and 14.2% children, respectively. Mean calcium concentration was 12.1 ± 2.0 mg/dl and PTH 91.8 ± 66.5 pmol/L. The sensitivity of preoperative imaging in parathyroid localization was 91.4%. Minimally invasive parathyroidectomy (MIP) was performed in 40% cases. Parathyroid adenoma was observed in 91.4% patients, whereas remaining had hyperplasia. Thirty-four percent suffered from Hungry bone syndrome in postoperative period. The cure rate following primary surgery was 97%. One child with persistent PHPT had successful re-operation. Median follow-up was 5 (1-17) years, and no recurrence or familial disease was revealed during this period. CONCLUSION: Majority of pediatric patients present with symptomatic PHPT. Despite relatively high incidence of familial disease select pediatric patients can undergo successful MIP.

Hereditary Medullary Thyroid Carcinoma: Genotype, Phenotype and Outcomes in a North Indian Cohort.

BACKGROUND: Aggressiveness of hereditary medullary thyroid carcinoma (hMTC) has been conventionally described to correlate with American Thyroid Association (ATA) risk groups based on RET mutations. Recent evidence increasingly contradicts this notion. We studied the RET genotype and its correlation with disease phenotype and survival outcomes in a cohort of hMTC patients. METHODS: In a retrospective cohort of 55 hMTC patients from 23 families treated at a north Indian tertiary care institute over 15-years, RET genotype was correlated with disease phenotype (clinical, biochemical, and pathological attributes) and outcomes in terms of biochemical cure (normalization of serum calcitonin), structural cure, overall survival (OS) and disease specific survival (DSS). RESULTS: Forty-nine patients had Multiple Endocrine Neoplasia (MEN)-type 2A syndrome, 02 had MEN-2B, and 4 had familial MTC. Two patients belonged to highest ATA risk, 41 to high-risk, and 12 to moderate risk categories. Age of the patients or stage of disease at presentation did not differ significantly between the ATA risk groups. Though the baseline serum calcitonin was significantly higher in highest risk category, the biochemical cure rates were not significantly different. At a median follow up of 48 months (Inter-quartile range 18-84, range 12-192) structural cure rates in ATA moderate and high risk groups were significantly higher than highest risk group (p = 0.04). No significant difference in OS between the three ATA groups of hMTC among the patients who underwent surgical treatment was observed (p = 0.098). CONCLUSIONS: The ATA moderate and high risk groups have better structural cure rates compared to ATA highest risk group. The biochemical cure and overall survival rates did not significantly differ between ATA risk-groups, and were impacted by the disease stage at presentation. The current ATA risk-groups do not reliably predict the outcomes in terms of biochemical cure and survival in hMTC patients.

Assessment of iodine nutrition of schoolchildren in Gonda, India, indicates improvement and effectivity of salt iodisation.

OBJECTIVE: To study the total goitre rate (TGR), urinary iodine concentration (UIC) and salt iodine content among schoolchildren in a previously endemic area for severe iodine deficiency disorder (IDD). DESIGN: Cross-sectional epidemiological study. SETTING: The study was carried out in the Gonda district (sub-Himalayan region) of North India. PARTICIPANTS: Nine hundred and seventy-seven schoolchildren (6-12 years) were studied for parameters such as height, weight, UIC and salt iodine content. Thyroid volume (TV) was measured by ultrasonography to estimate TGR. RESULTS: The overall TGR in the study population was 2·8 % (95 % CI 1·8, 3·8). No significant difference in TGR was observed between boys and girls (3·5 % v. 1·9 %, P = 0·2). There was a non-significant trend of increasing TGR with age (P = 0·05). Median UIC was 157·1 µg/l (interquartile range: 94·5-244·9). At the time of the study, 97 % of salt sample were iodised and nearly 86 % of salt samples had iodine content higher than or equal to 15 part per million. Overall, TGR was significantly lower (2·8 % v. 31·0 %, P < 0·001), and median UIC was significantly higher (157·1 v. 100·0 µg/l, P < 0·05) than that reported in the same area in 2009. CONCLUSIONS: A marked improvement was seen in overall iodine nutrition in the Gonda district after three and a half decades of Universal Salt Iodisation (USI). To sustainably control IDD, USI and other programmes, such as health education, must be continuously implemented along with putting mechanisms to monitor the programme at regular intervals in place.

Development of a dual specific growth rate-based fed-batch process for production of recombinant human granulocyte colony-stimulating factor in Pichia pastoris.

A number of limitations exist for production of human granulocyte colony-stimulating factor (G-CSF) in Pichia pastoris. In this study, two different specific growth rates (0.015 h-1, 0.01 h-1) were used sequentially in the mixed substrate feeding period during methanol induction phase to enhance the G-CSF titer in the culture broth. Necessary parameters required for implementing the feeding strategy, such as specific product yield on biomass (YP/X) and maintenance coefficient (m) on glycerol, methanol, and sorbitol were estimated using continuous culture technique. Using this strategy, for the same volumetric productivity, about 20% increase in protein titer was achieved over that obtained from the run carried out at a single pre-set value of 0.015 h-1 alone. Thus, implementation of higher specific growth rate (0.015 h-1) set during initial stages of the methanol induction phase followed by a lower specific growth rate (0.01 h-1) helped in achieving increased protein titers.

Purified cellobiose dehydrogenase of Termitomyces sp. OE147 fuels cellulose degradation resulting in the release of reducing sugars.

Termitomyces sp. OE 147 is one of the active cellulose degraders in the ecosphere and produces large amount of cellobiose dehydrogenase (CDH) and ß-glucosidases when cultivated on cellulose. In order to investigate its effect on cellulose, a highly purified preparation of CDH was obtained from the culture supernatant of the fungus cultivated on cellulose. A combination of ultrafiltration, ion-exchange and gel-filtration chromatography was used to purify CDH by ∼172-fold to a high specific activity of ∼324 U/mg protein on lactose which was used for routine measurement of enzyme activity. The enzyme displayed a pH optimum of 5.0 and stability between pH 5.0 and 8.0 with maximum catalytic efficiency (kcat/Km) of 397 mM-1 s-1 on cellobiose. Incubation of microcrystalline cellulose with the purified CDH led to production of reducing sugars which was accelerated by the addition of FeCl3 during the early stages of incubation. A mass spectrometric analysis revealed fragmentation products of cellulose which were concluded to be cellodextrins, sugars, and corresponding aldonic acids suggesting that CDH can release reducing sugars in the absence of externally added lytic polysaccharide monooxygenases. Polymerized products of glucose were also detected at low intensity.

Modifications in the Kex2 P1' cleavage site in the α-MAT secretion signal lead to higher production of human granulocyte colony-stimulating factor in Pichia pastoris.

The human granulocyte colony-stimulating factor (G-CSF) is one of the hematopoietic growth factors administered for chemotherapy induced neutropenia and is currently produced through recombinant route in Escherichia coli. The methylotrophic unicellular yeast Pichia pastoris (syn. Komagataella phaffii) makes a good host for production of human therapeutics as the proteins are low-mannose glycosylated, disulfide bonded and correctly folded on their way to the cell exterior. Given the low level of production of G-CSF in P. pastoris, the present study examined modification of the Saccharomyces cerevisiae derived α-mating type secretory signal sequence to enhance its production. The substitution of Glu, at the P1' position of the Kex2 cleavage site, by Val/Ala led to extracellular production of ~ 60 mg/L of G-CSF in the extracellular medium. Production was further increased to ~ 100 mg/L by putting these mutations against rarely occurring methanol slow utilization P. pastoris X-33 host. Analysis of the modelled structure of the signal peptide indicated exposed loop structures, created by presence of Val/Ala, that favour cleavage by the Kex2 peptidase thereby leading to enhanced production of G-CSF. The conformational changes, induced on account of binding between the signal sequence and the cargo protein (G-CSF), also appear to play an important role in the final yield of the extracellular protein.

Changing paradigms in endoscopic thyroid surgery: A comparison between scarless-in-the-neck axillo-breast approach and totally scarless transoral approach.

BACKGROUND: To avoid cervical scar in thyroid surgery, various approaches have been proposed. The commonly used approach is combined axillo-breast approach (ABA). However, trans-orovestibular approach (TOVA) is getting popular. The aim of this study is to compare surgical outcomes of patients who underwent endoscopic hemithyroidectomy (EHT) by either ABA or TOVA. PATIENTS AND METHODS: This was a retrospective analysis of clinical data of patients who underwent EHT from January 2013 to December 2018. Patients were divided into two groups: Group A - through ABA and Group B - through TOVA. RESULTS: A total of eighty patients underwent EHT in Group A and 25 in Group B. In both groups, most patients were female (male: female = 1:4.7 in Group A and 1:7.33 in Group B, P = 0.515). In both groups, there was no difference in age (the mean age was 33.44 ± 10.44 years in Group A and 33.04 ± 14.01 years in Group B, P = 0.391) and in size of the nodule (Group A - 3.91 ± 1.17 cm and Group B - 3.6 ± 1.39 cm, P = 0.228). The operating time was significantly less in Group B (Group A - 152.25 ± 30.19 mins and Group B - 126.80 ± 22.94 mins, P ≤ 0.01). The post-operative hospital stay was significantly less in Group B (mean 3.17 ± 0.97 days in Group A and 2.24 ± 0.60 days in Group B, P ≤ 0.01). CONCLUSION: TOVA is associated with shorter operating time and hospital stay with comparable outcomes. Cosmetic outcome is excellent in TOVA, however requires further subjective evaluation.

Differential role of segments of α-mating factor secretion signal in Pichia pastoris towards granulocyte colony-stimulating factor emerging from a wild type or codon optimized copy of the gene.

BACKGROUND: The methylotrophic yeast, Pichia pastoris has been widely used for the production of human therapeutics, but production of granulocyte colony-stimulating factor (G-CSF) in this yeast is low.The work reported here aimed to improve the extracellular production of G-CSF by introducing mutations in the leader sequence and using a codon optimized copy of G-CSF. Bioinformatic analysis was carried out to propose an explanation for observed effect of mutations on extracellular G-CSF production. RESULTS: Mutations in the pro-region of the α-mating type (MAT) secretory signal, when placed next to a codon optimized (CO)-GCSF copy, specifically, the Δ57-70 type, led to highest G-CSF titre of 39.4 ± 1.4 mg/L. The enhanced effect of this deletion was also observed when it preceded the WT copy of the gene. Deletion of the 30-43 amino acids in the pro-peptide, fused with the wild type (WT)-GCSF copy, completely diminished G-CSF secretion, while no effect was observed when this deletion was in front of the CO-GCSF construct. Also, Matα:Δ47-49 deletion preceding the WT-GCSF dampened the secretion of this protein, while no effect was seen when this deletion preceded the CO-GCSF copy of the gene. This indicated that faster rates of translation (as achieved through codon optimization) could overcome the control exercised by these segments. The loss of secretion occurring due to Δ30-43 in the WT-GCSF was partially restored (by 60%) when the Δ57-70 was added. The effect of Δ47-49 segment in the WT-GCSF could also be partially restored (by 60%) by addition of Δ57-70 indicating the importance of the 47-49 region. A stimulatory effect of Δ57-70 was confirmed in the double deletion (Matα:Δ57-70;47-49) construct preceding the CO-GCSF. Secondary and tertiary structures, when predicted using I-TASSER, allowed to understand the relationship between structural changes and their impact on G-CSF secretion. The Δ57-70 amino acids form a major part of 3rd alpha-helix in the pre-pro peptide and its distortion increased the flexibility of the loop, thereby promoting its interaction with the cargo protein. A minimum loop length was found to be necessary for secretion. The strict control in the process of secretion appeared to be overcome by changing the secondary structures in the signal peptides. Such fine tuning can allow enhanced secretion of other therapeutics in this expression system. CONCLUSIONS: Among the different truncations (Matα:Δ57-70, Matα:Δ47-49, Matα:Δ30-43, Matα:Δ57-70;30-43, Matα:Δ57-70;47-49) in pro-peptide of α-MAT secretion signal, Matα:Δ57-70 fused to CO-GCSF, led to highest G-CSF titre as compared to other Matα truncations. On the other hand, Matα:Δ30-43 and Matα:Δ47-49 fused to the WT-GCSF dampened the secretion of this protein indicating important role of these segments in the secretion of the cargo protein.

Smartphone-Based Application for Tele-follow-up of Patients with Endocrine Disorders in Context of a LMIC: A Compliance, Satisfaction, Clinical Safety and Outcome Assessment.

BACKGROUND: Remote video consultations are increasingly used in clinical practice, and a lot of data are emerging on its feasibility and acceptability. This study aims to bring out qualitative and quantitative data which will enhance our understanding of strengths and limitations of this media in the setting of a low- and middle-income country (LMIC). MATERIALS AND METHODS: This was a prospective study conducted from January 2017 to May 2018, at the Department of Endocrine Surgery, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Lucknow, India. A total of 107 patients were selected who chose remote follow-up care through social media (WhatsApp). Data were analyzed on feasibility, clinical safety, satisfaction and economic burden. RESULTS: A total of 107 postoperative patients were followed up using a social media tool. A total of 396 tele-sessions were held. The average number of tele-consultations per patient was 3.7 (range 2-6). Reasons for tele-follow-up included: confirmation of histology report (n = 92), medication dosage adjustments (n = 148), wound evaluation (n = 102), reporting of serum TSH and serum calcium levels (n = 296) and medical fitness certificate (n = 13). Wound evaluation through tele-follow-up was on par with the outpatient department (OPD) follow-up as no patient had to report to OPD for wound infection. Satisfaction level was excellent in 55% of patients and very good in 25%. 20% of the patients reported an average satisfaction level. If all of these 107 patients would have come to our OPD follow-up, they would have traveled 613.2 miles (908 km) per patient on an average, apart from losing work hours. Average cost and workdays saved per visit were $78 and 5.4 days, respectively. CONCLUSION: Video consultation using social media tools is clinically safe and cost effective. Economic benefits far outweigh the risk of missing an adverse event, especially in the setting of low- and middle-income countries.

Endoscopic versus Conventional Thyroid Surgery: A Comparison of Quality of Life, Cosmetic Outcomes and Overall Patient Satisfaction with Treatment.

BACKGROUND: Our aim was to investigate whether the surgical approach, i.e. endoscopic (ET) versus conventional/open thyroidectomy (ConT), contributes towards the improvement in quality of life (QoL), cosmetic outcomes and overall patient satisfaction with treatment. METHODS: We conducted a prospective study (October 2016-August 2018) consisting of patients undergoing hemithyroidectomy for euthyroid, non-malignant solitary thyroid nodules (STNs). Patients were divided into two groups: Group I (ET)-41 patients and Group II (ConT)-52 patients. ET was performed via bilateral-axillo breast approach (BABA). A thyroid disease-specific questionnaire "ThyPRO-39hin" was used to evaluate pre- and post-operative QoL. Visual analogue scale (VAS) was used for post-operative pain. Cosmetic satisfaction, paraesthesia and overall satisfaction with treatment were evaluated using Likert-type verbal response scales. Pre- and post-operative scores (>6 months) were compared, and p value < 0.05 was considered significant. RESULTS: Mean age (p = 0.26), gender distribution (p = 0.07), mean tumour size (p = 0.74) and preoperative scores of QoL were comparable between the two groups. Post-operatively scores of symptom domain (p = 0.03), tiredness (p = 0.03), impaired social life (p = 0.03), cosmetic complaints (p = < 0.001) and overall QoL (p = < 0.001) were significantly better in Group I. Also, post-operative pain perception at first follow-up visit (p = 0.001) was lower in patients undergoing ET/Group I, with higher scar satisfaction scores (p = < 0.001) and overall satisfaction with treatment (p = < 0.001). Post-operative paraesthesia perception was comparable (p = 0.06) amongst the two groups. CONCLUSION: In this cohort study, patients undergoing endoscopic thyroid surgery reported superior post-operative QoL, cosmetic and overall satisfaction with treatment as compared to conventional/open thyroidectomy.

Low-cost Fluorescein as an Alternative to Radio-colloid for Sentinel Lymph Node Biopsy-a Prospective Validation Study in Early Breast Cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) using radio-pharmaceutical (RP) and a blue dye is gold standard for axillary staging in clinically node-negative early breast cancer. High costs and limited availability of RP and/or gamma probe are major deterrents in performing SLNB in developing countries. Fluorescence-guided SLNB can obviate the need for RP and gamma probe. Fluorescein is an inexpensive fluorescent lymphatic tracer. In this study, we compared SLN identification rate (SLN-IR) and false negative rates (FNR) of fluorescein-guided SLNB and radio-guided SLNB using 99mTc-Sulfur-colloid, in isolation, or in combination with methylene blue dye (MBD). METHODS: Sixty-five cN0 early and large operable breast cancer patients underwent validation SLNB using fluorescein (and blue LED light), 99mTc-Sulfur-colloid (and gamma probe) and MBD. Inj Fluorescein 4% was injected, 1 ml each peri-tumoral and sub-areolar five minutes before axillary incision. Axillary dissection was performed irrespective of SLNB histology. The SLN-IR and FNR with various tracers and their combinations were compared. RESULTS: The mean number of SLNs identified was 3.5 ± 1.8 (range 1-6). The SLN-IR using RP alone was 94%, fluorescein alone was 92%, and MBD alone was 82%. The SLN-IR using fluorescein plus MBD combination was 95.4%, compared to 97% using MBD plus RP combination. FNR was 6.3% using fluorescein plus MBD, as well as RP plus MBD combinations. CONCLUSIONS: SLN-IR of > 90% and SLN-FNR of < 10% using fluorescein plus MBD combination are in acceptable range, and are comparable to that of RP plus MBD combination. Fluorescein can replace RP for performing SLNB, in combination with MBD.