High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.

Genome Dynamics of Vibrio cholerae Isolates Linked to Seasonal Outbreaks of Cholera in Dhaka, Bangladesh.

The temporal switching of serotypes from serotype Ogawa to Inaba and back to Ogawa was identified in Vibrio cholerae O1, which was responsible for seasonal outbreaks of cholera in Dhaka during the period 2015 to 2018. In order to delineate the factors responsible for this serotype transition, we performed whole-genome sequencing (WGS) of V. cholerae O1 multidrug-resistant strains belonging to both the serotypes that were isolated during this interval where the emergence and subsequent reduction of the Inaba serotype occurred. The whole-genome-based phylogenetic analysis revealed clonal expansion of the Inaba isolates mainly responsible for the peaks of infection during 2016 to 2017 and that they might have evolved from the prevailing Ogawa strains in 2015 which coclustered with them. Furthermore, the wbeT gene in these Inaba serotype isolates was inactivated due to insertion of a transposable element at the same position signifying the clonal expansion. Also, V. cholerae isolates in the Inaba serotype dominant clade mainly contained classical ctxB allele and revealed differences in the genetic composition of Vibrioseventh pandemic island II (VSP-II) and the SXT integrative and conjugative element (SXT-ICE) compared to those of Ogawa serotype strains which remerged in 2018. The variable presence of phage-inducible chromosomal island-like element 1 (PLE1) was also noted in the isolates of the Inaba serotype dominant clade. The detailed genomic characterization of the sequenced isolates has shed light on the forces which could be responsible for the periodic changes in serotypes of V. cholerae and has also highlighted the need to analyze the mobilome in greater detail to obtain insights into the mechanisms behind serotype switching.IMPORTANCE The switching of serotype from Ogawa to Inaba and back to Ogawa has been observed temporally in Vibrio cholerae O1, which is responsible for endemic cholera in Bangladesh. The serospecificity is key for effective intervention and for preventing cholera, a deadly disease that continues to cause significant morbidity and mortality worldwide. In the present study, WGS of V. cholerae allowed us to better understand the factors associated with the serotype switching events observed during 2015 to 2018. Genomic data analysis of strains isolated during this interval highlighted variations in the genes ctxB, tcpA, and rtxA and also identified significant differences in the genetic content of the mobilome, which included key elements such as SXT ICE, VSP-II, and PLE. Our results indicate that selective forces such as antibiotic resistance and phage resistance might contribute to the clonal expansion and predominance of a particular V. cholerae serotype responsible for an outbreak.

Clinicoradiologic Profile and Outcome of Children With Tubercular Meningitis in a Tertiary Care Hospital in Bangladesh.

BACKGROUND: Children are most vulnerable to tubercular meningitis. Neuroimaging is an important initial investigation in tubercular meningitis. OBJECTIVE: This study was done to describe the clinical profile, neuroimaging changes, and clinical outcome in children with tubercular meningitis. METHODOLOGY: This was an observational cohort study on children with tubercular meningitis, between January 2012 and June 2018. Tubercular meningitis was diagnosed on the basis of clinical criteria, cerebrospinal fluid analysis, neuroimaging, and response to antitubercular drug treatment. Preferably magnetic resonance imaging (MRI) with contrast was done. RESULT: Out of 79 pediatric patients, 17 patients were lost during follow-up; thus, a total of 62 patients were studied. Mean age at presentation was 7.040 (±3.99 SD) year, 51.6% children were male. Rural children were more affected. Twenty eight (45.2 patients had contact with a person with tuberculosis. Only 3 (4.8%) patients presented within 10 days of duration of illness. Most of the cases (67.7%) were in stage 2 at the time of diagnosis. The most common clinical feature was fever, seizure, and signs of meningeal irritation (all present in 12.9%). In neuroimaging most common findings were tuberculoma (50%), hydrocephalus (54.8%), and basal meningeal enhancement (33.8%). Regarding outcome, 6 (9.67%) patients expired and 47 (75%) patients had sequelae. The most common complications were hydrocephalus (30.64%) and intellectual disability (12.9 ). Hydrocephalus was the most common neuroimaging finding among the patients who expired (33%). CONCLUSION: Hydrocephalus is the most common neuroimaging finding. Normal neuroimaging is associated with good outcome whereas all the patients who died had abnormal neuroimaging.

Mutation Spectrum in TPO Gene of Bangladeshi Patients with Thyroid Dyshormonogenesis and Analysis of the Effects of Different Mutations on the Structural Features and Functions of TPO Protein through In Silico Approach.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.

Death audit in the neonatal ICU of a tertiary care hospital in Bangladesh: a retrospective chart review.

BACKGROUND: Expectant reduction of neonatal mortality and formulation of preventive strategies can only be achieved by analysis of risk factors in a particular setting. This study aimed to document incidence of neonatal death and to analyze the risk factors associated with neonatal death. METHODS: This retrospective study was carried out in department of Neonatology, Bangabandhu Sheikh Mujib Medical University (BSMMU) over a 12-month period from January to December 2015. The newborns that died within 28 d of life were defined as "Cases" and "Control" were the surviving newborn discharged to home as healthy. Two birth weight and gestational age matched controls were taken for each case. Maternal, obstetric, and newborn characteristics were analyzed between both the groups. Data analysis was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL). A probability of < .05 was considered statistically significant. The strength of association was determined by calculating odds ratio and their 95% confidence intervals (CIs). RESULTS: During the study period, the proportion of death was 9.6% (64/612). Both in Chi-square analysis and in logistic regression analysis, less than four antenatal visits (odds ratio (OR) 2.78; 95% CI: 1.23-6.28, p = .014) and sepsis (OR 2.37; 95% CI: 1.07-5.26, p = .034) were found to be independent risk factors for deaths, whereas LUCS found to be protective for deaths (OR 0.40; 95% CI: 0.19-0.83, p = .015). CONCLUSION: In conclusion, less than four antenatal visits and presence of sepsis were found to be independent risk factors whereas LUCS protective of newborn death.

Neonatal Glycemic Status of Infants of Diabetic Mothers in a Tertiary Care Hospital.

BACKGROUND: Diabetes is a common medical complication during pregnancy that results in significant neonatal morbidities. In infants of diabetic mothers (IDMs), hypoglycemia is a common complication. OBJECTIVE: To study the neonatal hypoglycemia in IDMs in a tertiary care hospital. SETTINGS AND DESIGN: A cross-sectional study was done in postnatal ward in Bangladesh Institute of Research and Rehabilitation in Diabetic, Endocrine and Metabolic Disorders from January to December 2009. SUBJECTS AND METHODS: The data of IDMs were collected from postnatal ward. All IDMs delivered during this period staying in postnatal ward were included in this study. The outcomes were compared between the hypoglycemic and normoglycemic IDMs and between gestational diabetes mellitus (GDM) and pre-GDM in hypoglycemic group using Chi-square test and Fisher's exact test. The data analysis was performed with Epi-enfo7 software. Statistical significance was set at P < 0.05. RESULTS: A total of 363 IDMs were included in this study. Hypoglycemia developed in 38.3% IDMs and 43.2% mothers of hypoglycemic IDMs had GDM and 56.8% had pre-GDM. Duration of maternal diabetes (P = 0.04) and large for gestational age (P = 0.0001) were associated with hypoglycemia. Multigravidae (82.2% vs 68.3%, P = 0.03), prolonged duration of maternal diabetes (45.46 weeks vs 3.23 weeks, P = 0.00001), preterm babies (48.1% vs 28.3% P = 0.009), and control of diabetes by insulin (81% vs 46.7%, P = 0.001) were more in pre-GDM, and statistically significant. About 85% IDMs developed hypoglycemia within 6 h of birth (P-value 0.00001) and majority (68%) were at 2 h of age. Forty percent of hypoglycemic IDMs from postnatal ward were admitted in special care baby unit. CONCLUSION: Hypoglycemia observed in 38.3% IDMs and developed within 6 h of age and maximum were at 2 h. Early recognition and appropriate intervention are needed in IDMs.

Clinical profile and pattern of congenital heart disease in infants of diabetic mother and infants of non-diabetic mother at a tertiary care hospital.

OBJECTIVES: To assess the clinical manifestations and pattern of congenital heart diseases (CHD) in infants of diabetic mothers (IDMs) and infants of non-diabetic mothers. METHODS: A prospective cross sectional study was carried out at tertiary care center over a period of thirty months. All neonates were included in this study if CHD were identified by echocardiography within this study period. Chi-square test and Fisher's exact test were used to compare between groups. RESULT: Out of 62 neonates, IDMs and non-IDMs were equal in number, male and female ratio was 3 : 2, and majority were delivered by cesarean section (85.5%). Mean gestational age was 35 weeks, 62.3% were preterm, and 37.7% were term. Common clinical presentations were cardiac murmur (61%), and respiratory distress (43.5%). Less common presentations were low SaO2 (27.4%), tachycardia (24.2%), and cyanosis (24.2%). Common combination of presentations were murmur and tachypnea (38.7%); cyanosis and low SaO2 (24.19%); and low SaO2 and tachypnea 15(24.19). The common echocardiographic findings were atrial septal defect (ASD; 72.6%), patent ductus arteriosus (PDA; 45.2%), and ventricular septal defect (VSD; 25.8%). ASD (77.4% vs 67.7%) and VSD (35.5% vs 16.1%) were more in IDMs, and PDA (48.4% vs 41.9%) was more in non-IDMs. These differences were not significant between groups. CONCLUSION: Clinical presentation and echocardiographic findings of CHD were similar in IDMs and non-IDMs.

Orexin gene transfer into zona incerta neurons suppresses muscle paralysis in narcoleptic mice.

Cataplexy, a sudden unexpected muscle paralysis, is a debilitating symptom of the neurodegenerative sleep disorder, narcolepsy. During these attacks, the person is paralyzed, but fully conscious and aware of their surroundings. To identify potential neurons that might serve as surrogate orexin neurons to suppress such attacks, the gene for orexin (hypocretin), a peptide lost in most human narcoleptics, was delivered into the brains of the orexin-ataxin-3 transgenic mouse model of human narcolepsy. Three weeks after the recombinant adenoassociated virus (rAAV)-mediated orexin gene transfer, sleep-wake behavior was assessed. rAAV-orexin gene delivery into neurons of the zona incerta (ZI), or the lateral hypothalamus (LH) blocked cataplexy. Orexin gene transfer into the striatum or in the melanin-concentrating hormone neurons in the ZI or LH had no such effect, indicating site specificity. In transgenic mice lacking orexin neurons but given rAAV-orexin, detectable levels of orexin-A were evident in the CSF, indicating release of the peptide from the surrogate neurons. Retrograde tracer studies showed that the amygdala innervates the ZI consistent with evidence that strong emotions trigger cataplexy. In turn, the ZI projects to the locus ceruleus, indicating that the ZI is part of a circuit that stabilizes motor tone. Our results indicate that these neurons might also be recruited to block the muscle paralysis in narcolepsy.

Hypocretin-2 saporin lesions of the ventrolateral periaquaductal gray (vlPAG) increase REM sleep in hypocretin knockout mice.

Ten years ago the sleep disorder narcolepsy was linked to the neuropeptide hypocretin (HCRT), also known as orexin. This disorder is characterized by excessive day time sleepiness, inappropriate triggering of rapid-eye movement (REM) sleep and cataplexy, which is a sudden loss of muscle tone during waking. It is still not known how HCRT regulates REM sleep or muscle tone since HCRT neurons are localized only in the lateral hypothalamus while REM sleep and muscle atonia are generated from the brainstem. To identify a potential neuronal circuit, the neurotoxin hypocretin-2-saporin (HCRT2-SAP) was used to lesion neurons in the ventral lateral periaquaductal gray (vlPAG). The first experiment utilized hypocretin knock-out (HCRT-ko) mice with the expectation that deletion of both HCRT and its target neurons would exacerbate narcoleptic symptoms. Indeed, HCRT-ko mice (n = 8) given the neurotoxin HCRT2-SAP (16.5 ng/23nl/sec each side) in the vlPAG had levels of REM sleep and sleep fragmentation that were considerably higher compared to HCRT-ko given saline (+39%; n = 7) or wildtype mice (+177%; n = 9). However, cataplexy attacks did not increase, nor were levels of wake or non-REM sleep changed. Experiment 2 determined the effects in mice where HCRT was present but the downstream target neurons in the vlPAG were deleted by the neurotoxin. This experiment utilized an FVB-transgenic strain of mice where eGFP identifies GABA neurons. We verified this and also determined that eGFP neurons were immunopositive for the HCRT-2 receptor. vlPAG lesions in these mice increased REM sleep (+79% versus saline controls) and it was significantly correlated (r = 0.89) with loss of eGFP neurons. These results identify the vlPAG as one site that loses its inhibitory control over REM sleep, but does not cause cataplexy, as a result of hypocretin deficiency.

Orexin (hypocretin) gene transfer diminishes narcoleptic sleep behavior in mice.

Gene transfer has proven to be an effective neurobiological tool in a number of neurodegenerative diseases, but it is not known if it can correct a sleep disorder. Narcolepsy is a neurodegenerative sleep disorder linked to the loss of neurons containing the neuropeptide orexin, also known as hypocretin. Here, a replication-defective herpes simplex virus-1 amplicon-based vector was constructed to transfer the gene for mouse prepro-orexin into mice with a genetic deletion of the orexin gene. After in vitro tests confirmed successful gene transfer into cells, the gene vector was delivered to the lateral hypothalamus of orexin knockout (KO) mice where the orexin peptide was robustly expressed in the somata and processes of numerous neurons, and the peptide product was detected in the cerebrospinal fluid. During the 4-day life-span of the vector the incidence of cataplexy declined by 60%, and the levels of rapid eye movement sleep during the second half of the night were similar to levels in wild-type mice, indicating that narcoleptic sleep-wake behavior in orexin KO mice can be improved by targeted gene transfer.

Entrainment of temperature and activity rhythms to restricted feeding in orexin knock out mice.

Ablation of the SCN, an established circadian clock, does not abolish food entrainment, suggesting that the food-entrainable oscillator (FEO) must lie outside the SCN. Typically, animals show anticipatory locomotor activity and rise in core body temperature under the influence of the FEO. Signals from the FEO would, therefore, converge onto arousal neurons so that the animal might forage for food. In the present study, we investigate whether the neuropeptide orexin, which has been linked to arousal, might transduce the arousal signal. Orexin-knockout (orexin-KO) and wildtype (WT) mice (both C57BL/6J derived) were implanted with MiniMitter transmitters that recorded core body temperature and activity (12 h LD cycle). After a week of ad-libitum feeding, the mice were given access to food for 4 h (ZT 4-8) for nine days followed by 2-days of fasting. When orexin-KO mice were placed in a restricted feeding schedule, both core body temperature and activity entrained to the feeding schedule. In these mice gross locomotor activity was severely blunted during the nine day period of restricted feeding (-79.4+/-6.3%) from the WT, but they showed an increase in core body temperature in anticipation to the meal time similar to the WT mice. There was no difference in the amount of food intake between the genotypes. We conclude that orexin is not required for entrainment of activity and temperature to a restricted feeding schedule, but is required for the robust expression of gross locomotor activity in anticipation of the scheduled feeding.

Sleep deprivation-induced protein changes in basal forebrain: implications for synaptic plasticity.

The need to sleep is universal and lack of sleep often results in decreases in alertness and cognitive function. Data suggest that sleep-related mechanisms and deficits resulting from loss of sleep are associated anatomically with the basal forebrain. Long-term effects of sleep deprivation, those lasting a day or more, likely require transcriptional changes leading to changes in protein synthesis, whereas short-term effects may be mediated by rapid changes in the functional status of proteins. To understand sleep deprivation-induced changes in proteins in the wake-promoting area of the basal forebrain in rat, proteomic analysis was carried out by a combination of 2D gel electrophoresis to separate and visualize proteins and matrix-assisted laser desorption/ionization time-of flight mass spectrometry for protein identification. Among 969 protein spots that were compared, 89 spots showed more than a twofold difference between 6-hr sleep-deprived rats and undisturbed sleeping controls. We have identified 11 of these proteins to be either cytoskeletal or associated with synapses. The changes in four of these proteins were analyzed further by Western blots of 1D and 2D. Two proteins associated with the cytoskeleton, tubulin and GAP43, show posttranslational modifications. Increased tyrosination of alpha tubulin isoforms and increased phosphorylation of GAP43 was observed after 6-hr sleep deprivation when compared to that in sleeping controls. The synaptic protein synaptosomal-associated protein-25 (SNAP25) is decreased whereas amphiphysin is phosphorylated after sleep deprivation. These changes in proteins in the basal forebrain during short-term sleep deprivation are suggestive of changes in the substrate for neuronal transmission and plasticity.