Comprehensive molecular analysis identifies eight novel variants in XY females with disorders of sex development.

Disorders of sex development (DSD) are a group of clinical conditions with variable presentation and genetic background. Females with or without development of secondary sexual characters and presenting with primary amenorrhea (PA) and a 46,XY karyotype are one of the classified groups in DSD. In this study, we aimed to determine the genetic mutations in 25 females with PA and a 46,XY karyotype to show correlations with their phenotypes. Routine Sanger sequencing with candidate genes like SRY, AR, SRD5A2, and SF1, which are mainly responsible for 46,XY DSD in adolescent females, was performed. In a cohort of 25 patients of PA with 46,XY DSD, where routine Sanger sequencing failed to detect the mutations, next-generation sequencing of a targeted gene panel with 81 genes was used for the molecular diagnosis. The targeted sequencing identified a total of 21 mutations including 8 novel variants in 20 out of 25 patients with DSD. The most frequently identified mutations in our series were in AR (36%), followed by SRD5A2 (20%), SF1 (12%), DHX37 (4%), HSD17B3 (4%), and DMRT2 (4%). We could not find any mutation in the DSD-related genes in five (20%) patients due to complex molecular mechanisms in 46,XY DSD, highlighting the possibility of new DSD genes which are yet to be discovered in these disorders. In conclusion, genetic testing, including cytogenetics and molecular genetics, is important for the diagnosis and management of 46,XY DSD cases.

Role of solution reconstruction in hypersonic viscous computations using a sharp interface immersed boundary method.

This work discusses the development of a sharp interface immersed boundary (IB) method for viscous compressible flows and its assessment for accurate computations of wall shear and heat fluxes in hypersonic flows. The IB method is implemented in an unstructured Cartesian finite-volume (FV) framework and resolves the geometric interface sharply on the nonconformal mesh through direct imposition of boundary conditions employing a local reconstruction approach. The efficacy of the IB-FV solver is investigated for canonical high-speed viscous flows over a range of Mach numbers. The numerical results indicate that the surface pressure and shear stress distributions are computed with reasonable accuracy, whereas surface heat fluxes for aerodynamically blunt configurations are underpredicted. Employing a set of carefully designed experiments and simple diagnostic tools, we probe the possible causes for the underprediction in heat flux. We show that there exist two sources of error-one due to grid resolution and the other due to solution reconstruction, with the latter being more prominent and responsible for the observed underprediction in heat fluxes. Studies reveal that the heat flux estimates are sensitive to the choice of temperature reconstruction and linear interpolations could lead to poor estimates of heat flux. Our investigations conclusively point out the fact that existing polynomial-based reconstruction approaches for sharp interface IB techniques are not necessarily adequate for heat transfer predictions in high Reynolds number hypersonic flows.

Machinability Analysis and Optimization in Wire EDM of Medical Grade NiTiNOL Memory Alloy.

NiTiNOL (Nickel-Titanium) shape memory alloys (SMAs) are ideal replacements for titanium alloys used in bio-medical applications because of their superior properties like shape memory and super elasticity. The machining of NiTiNOL alloy is challenging, as it is a difficult to cut material. Hence, in the current research the experimental studies on machinability aspects of medical grade NiTiNOL SMA during wire electric discharge machining (WEDM) using zinc coated brass wire as electrode material have been carried out. Pulse time (Ton), pause time (Toff), wire feed (WF), and servo voltage (SV) are chosen as varying input process variables and the effects of their combinational values on output responses such as surface roughness (SR), material removal rate (MRR), and tool wear rate (TWR) are studied through response surface methodology (RSM) based developed models. Modified differential evolution (MDE) optimization technique has been developed and the convergence curve of the same has been compared with the results of differential evolution (DE) technique. Scanning electron microscopy (SEM) and energy dispersive X-ray spectrography (EDS) analysis are carried out to study the surface morphology of the machined alloy. SV is found to be more influential process parameter for achieving better MRR with minimal SR and TWR, followed by Ton, Toff, and WF. The WF has good impact on reduced SR and TWR responses and found to be least significant in maximizing MRR.

High-temperature hypersonic Laval nozzle for non-LTE cavity ringdown spectroscopy.

A small dimension Laval nozzle connected to a compact high enthalpy source equipped with cavity ringdown spectroscopy (CRDS) is used to produce vibrationally hot and rotationally cold high-resolution infrared spectra of polyatomic molecules in the 1.67 µm region. The Laval nozzle was machined in isostatic graphite, which is capable of withstanding high stagnation temperatures. It is characterized by a throat diameter of 2 mm and an exit diameter of 24 mm. It was designed to operate with argon heated up to 2000 K and to produce a quasi-unidirectional flow to reduce the Doppler effect responsible for line broadening. The hypersonic flow was characterized using computational fluid dynamics simulations, Pitot measurements, and CRDS. A Mach number evolving from 10 at the nozzle exit up to 18.3 before the occurrence of a first oblique shock wave was measured. Two different gases, carbon monoxide (CO) and methane (CH4), were used as test molecules. Vibrational (Tvib) and rotational (Trot) temperatures were extracted from the recorded infrared spectrum, leading to Tvib = 1346 ± 52 K and Trot = 12 ± 1 K for CO. A rotational temperature of 30 ± 3 K was measured for CH4, while two vibrational temperatures were necessary to reproduce the observed intensities. The population distribution between vibrational polyads was correctly described with Tvib I=894±47 K, while the population distribution within a given polyad (namely, the dyad or the pentad) was modeled correctly by Tvib II=54±4 K, testifying to a more rapid vibrational relaxation between the vibrational energy levels constituting a polyad.

Chromosomal Aberrations in Primary Amenorrhea: A Retrospective Study.

OBJECTIVES: The aim of this study was to estimate the frequency of chromosomal abnormalities and establish the association with clinical of factors such as secondary sexual characters and gonad development in primary amenorrhea (PA). STUDY DESIGN: The study was carried out in a large cohort of PA. The chromosomal aberrations were correlated with secondary sexual characters and anatomical abnormalities. MATERIALS AND METHODS: The data of 490 cases of PA were collected retrospectively. The chromosomal preparations were done from the peripheral blood and subjected to giemsa-trypsin-giemsa banding and karyotyped according to the International System of Human Cytogenetic Nomenclature 2013. The fluorescence in situ hybridization was carried out using centromeric and whole painting probes for X and Y chromosome. STATISTICAL ANALYSIS: Statistical analysis of the data was performed using online version of social science statistics software. RESULTS: A high frequency of abnormal uterus (81.9%) and ovaries (86.7%) were detected in our study. A total of 121 (24.7%) cases were identified with abnormal karyotype. The numerical chromosomal abnormalities were identified in 53 (43.8%) cases while structural abnormalities were identified in 32 (26.4%) cases. The XY karyotype was detected in 29.8% females with PA. The PA individuals with anatomical abnormalities (84.3%) had a high frequency (24.6%) of chromosomal aberrations. CONCLUSIONS: The present study concluded that cytogenetics plays an important role in precise diagnosis which helps in the management of PA. The cytogenetic analysis should be carried out to know the genetic basis of PA.

In-gel and OFFGEL-based proteomic approach for authentication of meat species from minced meat and meat products.

BACKGROUND: Fraudulent mislabelling of processed meat products on a global scale that cannot be detected using conventional techniques necessitates sensitive, robust and accurate methods of meat authentication to ensure food safety and public health. In the present study, we developed an in-gel (two-dimensional gel electrophoresis, 2DE) and OFFGEL-based proteomic method for authenticating raw and cooked water buffalo (Bubalus bubalis), sheep (Ovis aries) and goat (Caprus hircus) meat and their mixes. RESULTS: The matrix-assisted liquid desorption/ionization time-of-flight mass spectrometric analysis of proteins separated using 2DE or OFFGEL electrophoresis delineated species-specific peptide biomarkers derived from myosin light chain 1 and 2 (MLC1 and MLC2) of buffalo-sheep-goat meat mix in definite proportions at 98:1:1, 99:0.5:0.5 and 99.8:0.1:0.1 that were found stable to resist thermal processing. In-gel and OFFGEL-based proteomic approaches are efficient in authenticating meat mixes spiked at minimum 1.0% and 0.1% levels, respectively, in triple meat mix for both raw and cooked samples. CONCLUSIONS: The study demonstrated that authentication of meat from a complex mix of three closely related species requires identification of more than one species-specific peptide due to close similarity between their amino acid sequences. © 2017 Society of Chemical Industry.

OFFGEL electrophoresis and tandem mass spectrometry approach compared with DNA-based PCR method for authentication of meat species from raw and cooked ground meat mixtures containing cattle meat, water buffalo meat and sheep meat.

The present study compared the accuracy of an OFFGEL electrophoresis and tandem mass spectrometry-based proteomic approach with a DNA-based method for meat species identification from raw and cooked ground meat mixes containing cattle, water buffalo and sheep meat. The proteomic approach involved the separation of myofibrillar proteins using OFFGEL electrophoresis, SDS-PAGE and protein identification by MALDI-TOF MS. Species-specific peptides derived from myosin light chain-1 and 2 were identified for authenticating buffalo meat spiked at a minimum 0.5% level in sheep meat with high confidence. Relative quantification of buffalo meat mixed with sheep meat was done by quantitative label-free mass spectrometry using UPLC-QTOF and PLGS search engine to substantiate the confidence level of the data. In the DNA-based method, PCR amplification of mitochondrial D loop gene using species specific primers found 226bp and 126bp product amplicons for buffalo and cattle meat, respectively. The method was efficient in detecting a minimum of 0.5% and 1.0% when buffalo meat was spiked with cattle meat in raw and cooked meat mixes.

Effect of sous vide processing on physicochemical, ultrastructural, microbial and sensory changes in vacuum packaged chicken sausages.

The processing of sous vide chicken sausages was optimized under vacuum packaging condition and cooking at 100 ℃ for 30 min (SV30), 60 min (SV60) and 120 min (SV120) and compared with aerobically cooked control at 100 ℃ for 30 min. Sous vide processing of chicken sausages (SV30) produced higher (p < 0.05) cooking yield, Hunterlab a* values and sensory attributes without affecting proximate composition and shear force values relative to control. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis and scanning electron microscopy results revealed no significant changes in protein quality and emulsion ultra-structure due to SV30 processing relative to control sausages. Sous vide processing of chicken sausages enriched with rosemary diterpene phenols retained the freshness and quality up to 120 days during storage at 4 ± 1 ℃ relative to control sausages that were spoiled on 20th day. Lipid oxidation and microbial growth remained below the spoilage levels for all the SV-processed sausages throughout the storage and addition of rosemary diterpene mixture at 0.02% v/w reduced the microbial growth and improved (p < 0.05) the sensory attributes. Our results demonstrate that sous vide processing minimizes lipid oxidation and microbial growth of chicken sausages with improved product quality and shelf-life at 4 ± 1 ℃.

Proteomic based approach for characterizing 4-hydroxy-2-nonenal induced oxidation of buffalo (Bubalus bubalis) and goat (Capra hircus) meat myoglobins.

BACKGROUND: Myoglobin (Mb) is a sarcoplasmic heme protein primarily responsible for meat color and its chemistry is species specific. 4-hydroxy-2-nonenal (HNE) is a cytotoxic lipid derived aldehyde detected in meat and was reported to covalently adduct with nucleophilic histidine residues of Mb and predispose it to greater oxidation. However, no literature is available on characterization of lipid oxidation induced oxidation of Indian water buffalo (Bubalus bubalis) and goat (Capra hircus) myoglobins. METHODS: Present study characterize the Mb extracted from water buffalo and goat cardiac muscles using two-dimensional gel electrophoresis (2DE), OFFGEL electrophoresis and mass spectrometry (MS). Purified buffalo and goat bright red oxymyoglobin were reacted with HNE in-vitro at physiological pH (7.4) and temperature (37 °C) conditions and the formation of oxidised brown metmyoglobin was measured. The Mb-HNE adducts were detected using MALDI-TOF MS, whereas specific sites of adduction was determined using ESI-QTOF MS/MS. RESULTS: Purified buffalo and goat Mb samples revealed a molecular mass of 17,043.6 and 16,899.9 Daltons, respectively. The 2DE analysis exhibited 65 (sarcoplasmic protein extract) and 6 (pure Mb) differentially expressed (P < 0.05) protein spots between buffalo and goat samples. OFFGEL electrophoresis revealed an isoelectric point of 6.77 and 7.35 respectively, for buffalo and goat Mb's. In-vitro incubation of HNE with bright red buffalo and goat oxymyoglobin's at pH 7.4 and 37 °C resulted in pronounced (P < 0.05) oxidation and formation of brown metmyoglobin. MALDI-TOF MS analysis of Mb-HNE reaction mix revealed covalent binding (via Michael addition) of 3 and 5 molecules of HNE with buffalo and goat Oxy-Mb's, respectively. ESI-QTOF MS/MS identified seven and nine histidine (HIS) residues of Mb that were readily adducted by HNE in buffalo and goat, respectively. CONCLUSION: The study demonstrated better redox stability of buffalo Mb than goat Mb. Our findings confirm the hypothesis that relative effect of HNE was greater for Mb's with 12 ± 1 HIS residues than Mb's with 9 HIS residues and helps meat processors in developing species-specific processing strategies to reduce the color variability.

Independent Mobility Achieved through a Wireless Brain-Machine Interface.

Individuals with tetraplegia lack independent mobility, making them highly dependent on others to move from one place to another. Here, we describe how two macaques were able to use a wireless integrated system to control a robotic platform, over which they were sitting, to achieve independent mobility using the neuronal activity in their motor cortices. The activity of populations of single neurons was recorded using multiple electrode arrays implanted in the arm region of primary motor cortex, and decoded to achieve brain control of the platform. We found that free-running brain control of the platform (which was not equipped with any machine intelligence) was fast and accurate, resembling the performance achieved using joystick control. The decoding algorithms can be trained in the absence of joystick movements, as would be required for use by tetraplegic individuals, demonstrating that the non-human primate model is a good pre-clinical model for developing such a cortically-controlled movement prosthetic. Interestingly, we found that the response properties of some neurons differed greatly depending on the mode of control (joystick or brain control), suggesting different roles for these neurons in encoding movement intention and movement execution. These results demonstrate that independent mobility can be achieved without first training on prescribed motor movements, opening the door for the implementation of this technology in persons with tetraplegia.

Influence of six medicinal herbs on collagenase-induced osteoarthritis in rats.

Medicinal herbs have been effectively used for their anti-inflammatory activity, but their exact role has not yet been documented in scientific literature for the management of Osteoarthritis (OA). Since Sida cordifolia L., Piper longum L., Zingiber officinale Rosc., Ricinus communis L., Vitex negundo L. and Tribulus terrestris L. have been widely used in traditional medicine for their anti-inflammatory activity, to evaluate anti-osteoarthritic activity of these herbs, we used a collagenase type II-induced osteoarthritis (CIOA) rat model. Arthritis was induced in wistar rats by intra-articular injection of collagenase type II. Powders of herbs were given orally for 20 days as a suspension in water (270 mg/kg b. wt.). The effects of the treatment in the rats were monitored by physiological parameters like body weight, knee diameter, paw retraction, paw volume, glycosaminoglycan (GAG) release, radiography and histopathology of knee joint. Selected herbs have significantly prevented body weight loss and knee swelling compared to arthritic control (CIOA). All test groups, including indomethacin (standard drug, 3 mg/kg), significantly reduced paw volume compared to CIOA. GAG release in the serum was significantly lowered in herb treated groups compared to indomethacin. The anterior posterior radiographs of S. cordifolia and P. longum treated groups showed a protective effect against OA. Histopathology revealed protection in the structure of the articular cartilage and in chondrocyte pathology as well as reduced clefting. Treatment with herbs has shown chondroid matrix within normal limits. From the results, we observed that S. cordifolia and P. longum possess potent anti-osteoarthritic activity.

Progranulin is a chemoattractant for microglia and stimulates their endocytic activity.

Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid ß1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid ß.

Chronic umbilical cord entanglements causing intrauterine fetal demise in the second trimester.

Entanglement of the umbilical cord with fetal body parts is known to occur in early pregnancy. This can potentially compromise the cord blood flow and cause fetal demise. We report 3 instances of intrauterine fetal deaths in the 2nd trimester of pregnancy with longstanding cord entanglement. The cord had left impressions of entanglement on the entrapped growing fetal part. Restricted movements of the fetus due to cord entanglement led to reduced spiraling of the umbilical cord. Our case series demonstrates that tight entanglement of fetal body parts by the umbilical cord can cause fetal demise in the 2nd trimester. This event is associated with a straight umbilical cord. Thus, the presence of reduced spiraling in intrauterine fetal demise warrants a search for possible cord entanglement along with established causes, such as chromosomal and congenital anomalies.

Exhaustive prediction of disease susceptibility to coding base changes in the human genome.

BACKGROUND: Single Nucleotide Polymorphisms (SNPs) are the most abundant form of genomic variation and can cause phenotypic differences between individuals, including diseases. Bases are subject to various levels of selection pressure, reflected in their inter-species conservation. RESULTS: We propose a method that is not dependant on transcription information to score each coding base in the human genome reflecting the disease probability associated with its mutation. Twelve factors likely to be associated with disease alleles were chosen as the input for a support vector machine prediction algorithm. The analysis yielded 83% sensitivity and 84% specificity in segregating disease like alleles as found in the Human Gene Mutation Database from non-disease like alleles as found in the Database of Single Nucleotide Polymorphisms. This algorithm was subsequently applied to each base within all known human genes, exhaustively confirming that interspecies conservation is the strongest factor for disease association. For each gene, the length normalized average disease potential score was calculated. Out of the 30 genes with the highest scores, 21 are directly associated with a disease. In contrast, out of the 30 genes with the lowest scores, only one is associated with a disease as found in published literature. The results strongly suggest that the highest scoring genes are enriched for those that might contribute to disease, if mutated. CONCLUSION: This method provides valuable information to researchers to identify sensitive positions in genes that have a high disease probability, enabling them to optimize experimental designs and interpret data emerging from genetic and epidemiological studies.