Biallelic variants in WARS1 cause a highly variable neurodevelopmental syndrome and implicate a critical exon for normal auditory function.

Aminoacyl-tRNA synthetases (ARSs) are essential enzymes for faithful assignment of amino acids to their cognate tRNA. Variants in ARS genes are frequently associated with clinically heterogeneous phenotypes in humans and follow both autosomal dominant or recessive inheritance patterns in many instances. Variants in tryptophanyl-tRNA synthetase 1 (WARS1) cause autosomal dominantly inherited distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. Presently, only one family with biallelic WARS1 variants has been described. We present three affected individuals from two families with biallelic variants (p.Met1? and p.(Asp419Asn)) in WARS1, showing varying severities of developmental delay and intellectual disability. Hearing impairment and microcephaly, as well as abnormalities of the brain, skeletal system, movement/gait, and behavior were variable features. Phenotyping of knocked down wars-1 in a Caenorhabditis elegans model showed depletion is associated with defects in germ cell development. A wars1 knockout vertebrate model recapitulates the human clinical phenotypes, confirms variant pathogenicity, and uncovers evidence implicating the p.Met1? variant as potentially impacting an exon critical for normal hearing. Together, our findings provide consolidating evidence for biallelic disruption of WARS1 as causal for an autosomal recessive neurodevelopmental syndrome and present a vertebrate model that recapitulates key phenotypes observed in patients.

Factors Associated with Absolute Neutrophil Count Dynamics and Docetaxel-Adryamicin-Cyclophosphamide (TAC) Chemotherapy Induced Neutropenia During Extended Filgrastim Administration in Breast Cancer Patients.

BACKGROUND: Myelosuppressive effects of chemotherapy for breast cancer treatment may trigger chemotherapy-induced neutropenia (CIN) and febrile neutropenia (FN). Filgrastim has been widely used as prophylaxis against CIN and FN. However despite filgrastim administration, some study showed FN still occur and cause patient vulnerability to infection. This study aims to evaluate factors associated with Absolute Neutrophil Count (ANC) dynamics and Docetaxel-Adryamicin-Cyclophosphamide (TAC) CIN during extended filgrastim administration in breast cancer patients. METHODS: Patients were selected among breast cancer in-patients who fulfilled the eligibility criteria. Patient characteristics data and ANC were collected. The entire patients received 5µg/kg/day filgrastim by subcutaneous injection 24 hours post-chemotherapy. ANC was monitored daily and filgrastim administration was stopped when ANC reached >10000/mm3 or 14 days of administration. Kruskall-Wallis test and Spearman Correlation test was performed to analyze ANC dynamics and CIN-related factors. RESULTS: This study included 42 breast cancer patients. Patient age median was 52 (31-70) years old. ANC nadir could be observed around 5-7 days after chemotherapy and FN occurred in two out of 38 grade 4 neutropenia patients (4.8%). Critical ANC lasted for 1 day, 2 days, and 3 days respectively in 9 (23.7%), 25 (65.8%) and 4 (10.5%) patients. There was no correlation between neutropenia and age. ANC slope and recovery duration did not show a significant difference. However, depth of nadir is inversely correlated with the duration of ANC recovery (>10000/mm3) and the duration during the peak on the 2nd day until reaching nadir both with fair strength, r = -0.489 and r = -0.438 (p <0.05), respectively. No sepsis incidence had manifested. CONCLUSION: CIN still occured in breast cancer patient receiving filgrastim primary prophylaxis regardless of age and neutropenia severity. Nadir as the lowest point of ANC should be noted as a pivotal milestone for ANC slope and recovery evaluation.

A novel nonsense variant in SLC24A4 causing a rare form of amelogenesis imperfecta in a Pakistani family.

BACKGROUND: Amelogenesis imperfecta (AI) is a highly heterogeneous group of hereditary developmental abnormalities which mainly affects the dental enamel during tooth development in terms of its thickness, structure, and composition. It appears both in syndromic as well as non-syndromic forms. In the affected individuals, the enamel is usually thin, soft, rough, brittle, pitted, chipped, and abraded, having reduced functional ability and aesthetics. It leads to severe complications in the patient, like early tooth loss, severe discomfort, pain, dental caries, chewing difficulties, and discoloration of teeth from yellow to yellowish-brown or creamy type. The study aimed to identify the disease-causing variant in a consanguineous family. METHODS: We recruited a consanguineous Pashtun family of Pakistani origin. Exome sequencing analysis was followed by Sanger sequencing to identify the pathogenic variant in this family. RESULTS: Clinical analysis revealed hypomaturation AI having generalized yellow-brown or creamy type of discoloration in affected members. We identified a novel nonsense sequence variant c.1192C > T (p.Gln398*) in exon-12 of SLC24A4 by using exome sequencing. Later, its co-segregation within the family was confirmed by Sanger sequencing. The human gene mutation database (HGMD, 2019) has a record of five pathogenic variants in SLC24A4, causing AI phenotype. CONCLUSION: This nonsense sequence variant c.1192C > T (p.Gln398*) is the sixth disease-causing variant in SLC24A4, which extends its mutation spectrum and confirms the role of this gene in the morphogenesis of human tooth enamel. The identified variant highlights the critical role of SLC24A4 in causing a rare AI type in humans.

Deleterious Variants in WNT10A, EDAR, and EDA Causing Isolated and Syndromic Tooth Agenesis: A Structural Perspective from Molecular Dynamics Simulations.

The dental abnormalities are the typical features of many ectodermal dysplasias along with congenital malformations of nails, skin, hair, and sweat glands. However, several reports of non-syndromic/isolated tooth agenesis have also been found in the literature. The characteristic features of hypohidrotic ectodermal dysplasia (HED) comprise of hypodontia/oligodontia, along with hypohidrosis/anhidrosis, and hypotrichosis. Pathogenic variants in EDA, EDAR, EDARADD, and TRAF6, cause the phenotypic expression of HED. Genetic alterations in EDA and WNT10A cause particularly non-syndromic/isolated oligodontia. In the current project, we recruited 57 patients of 17 genetic pedigrees (A-Q) from different geographic regions of the world, including Pakistan, Egypt, Saudi Arabia, and Syria. The molecular investigation of different syndromic and non-syndromic dental conditions, including hypodontia, oligodontia, generalized odontodysplasia, and dental crowding was carried out by using exome and Sanger sequencing. We have identified a novel missense variant (c.311G>A; p.Arg104His) in WNT10A in three oligodontia patients of family A, two novel sequence variants (c.207delinsTT, p.Gly70Trpfs*25 and c.1300T>G; p.Try434Gly) in EDAR in three patients of family B and four patients of family C, respectively. To better understand the structural and functional consequences of missense variants in WNT10A and EDAR on the stability of the proteins, we have performed extensive molecular dynamic (MD) simulations. We have also identified three previously reported pathogenic variants (c.1076T>C; p.Met359Thr), (c.1133C>T; p.Thr378Met) and (c.594_595insC; Gly201Argfs*39) in EDA in family D (four patients), E (two patients) and F (one patient), correspondingly. Presently, our data explain the genetic cause of 18 syndromic and non-syndromic tooth agenesis patients in six autosomal recessive and X-linked pedigrees (A-F), which expand the mutational spectrum of these unique clinical manifestations.

Brachyolmia, dental anomalies and short stature (DASS): Phenotype and genotype analyses of Egyptian and Pakistani patients.

Brachyolmia is a heterogeneous group of developmental disorders characterized by a short trunk, short stature, scoliosis, and generalized platyspondyly without significant deformities in the long bones. DASS (Dental Abnormalities and Short Stature), caused by alterations in the LTBP3 gene, was previously considered as a subtype of brachyolmia. The present study investigated three unrelated consanguineous families (A, B, C) with Brachyolmia and DASS from Egypt and Pakistan. In our Egyptian patients, we also observed hearing impairment. Exome sequencing was performed to determine the genetic causes of the diverse clinical conditions in the patients. Exome sequencing identified a novel homozygous splice acceptor site variant (LTBP3:c.3629-1G > T; p. ?) responsible for DASS phenotypes and a known homozygous missense variant (CABP2: c.590T > C; p.Ile197Thr) causing hearing impairment in the Egyptian patients. In addition, two previously reported homozygous frameshift variants (LTBP3:c.132delG; p.Pro45Argfs*25) and (LTBP3:c.2216delG; p.Gly739Alafs*7) were identified in Pakistani patients. This study emphasizes the vital role of LTBP3 in the axial skeleton and tooth morphogenesis and expands the mutational spectrum of LTBP3. We are reporting LTBP3 variants in seven patients of three families, majorly causing brachyolmia with dental and cardiac anomalies. Skeletal assessment documented short webbed neck, broad chest, evidences of mild long bones involvement, short distal phalanges, pes planus and osteopenic bone texture as additional associated findings expanding the clinical phenotype of DASS. The current study reveals that the hearing impairment phenotype in Egyptian patients of family A has a separate transmission mechanism independent of LTBP3.

Mechanism of Stone (Hardened Endocarp) Formation in Fruits: An Attempt toward Pitless Fruits, and Its Advantages and Disadvantages.

Stone (hardened endocarp) has a very important role in the continuity of plant life. Nature has gifted plants with various seed protection and dispersal strategies. Stone-fruit-bearing species have evolved a unique adaptation in which the seed is encased in an extremely hard wood-like shell called the stone. The lignification of the fruit endocarp layer produces the stone, a feature that separates drupes from other plants. Stone cells emerge from parenchyma cells after programmed cell death and the deposition of cellulose and lignin in the secondary cell wall. Generally, the deposition of lignin in primary cell walls is followed by secondary thickening of cell walls to form stone cells. This review article describes the molecular mechanisms and factors that influence the production of stone in the fruit. This is the first review article that describes the molecular mechanisms regulating stone (harden endocarp) formation in fruits. This article will help breeders understand the molecular and genetic basis for the stone formation in fruit, and this could lead to new and innovative directions to breed stoneless fruit cultivars in the future.

FhuA deletion variant Δ1-159 overexpression in inclusion bodies and refolding with Polyethylene-Poly(ethylene glycol) diblock copolymer.

Membrane protein isolation is a challenging problem. In fact especially their extraction from the respective membrane is difficult and often goes along with losses in yield. Usually expensive detergents are needed to extract the target protein from the membrane. Therefore finding an efficient overexpression and extraction method and an alternative to detergents is desirable. In this study we describe a new and fast method to express, extract and purify an engineered variant of the FhuA protein (FhuA Δ1-159) that acts as passive diffusion channel, using a diblock copolymer as an alternative to detergents like octyl-POE (n-octylpolyoxyethylene). The N-terminal leader sequence, facilitating the protein's transport to the outer membrane was deleted (FhuA Δ1-159 Δsignal), resulting in protein accumulation in easy to isolate inclusion bodies. Urea was used to solubilise the unfolded protein and dialysis against phosphate-buffer containing the commercially available diblock copolymer PE-PEG[Polyethylene-Poly(ethyleneglycol)] lead to protein refolding. Circular dichroism spectroscopy revealed a high ß-sheet percentage within the refolded protein secondary structure indicating the successful reconstitution of FhuA Δ1-159 Δsignal native state. Furthermore the channel functionality of FhuA Δ1-159 Δsignal was verified by measuring the in and out-flux through the protein when inserted into liposome membrane, using the HRP/TMB (HRP=Horse Radish Peroxidase, TMB=3,3',5,5'-tetramethylbenzidine) assay system.

Engineering of the E. coli outer membrane protein FhuA to overcome the hydrophobic mismatch in thick polymeric membranes.

BACKGROUND: Channel proteins like the engineered FhuA Δ1-159 often cannot insert into thick polymeric membranes due to a mismatch between the hydrophobic surface of the protein and the hydrophobic surface of the polymer membrane. To address this problem usually specific block copolymers are synthesized to facilitate protein insertion. Within this study in a reverse approach we match the protein to the polymer instead of matching the polymer to the protein. RESULTS: To increase the FhuA Δ1-159 hydrophobic surface by 1 nm, the last 5 amino acids of each of the 22 ß-sheets, prior to the more regular periplasmatic ß-turns, were doubled leading to an extended FhuA Δ1-159 (FhuA Δ1-159 Ext). The secondary structure prediction and CD spectroscopy indicate the ß-barrel folding of FhuA Δ1-159 Ext. The FhuA Δ1-159 Ext insertion and functionality within a nanocontainer polymeric membrane based on the triblock copolymer PIB(1000)-PEG(6000)-PIB(1000) (PIB = polyisobutylene, PEG = polyethyleneglycol) has been proven by kinetic analysis using the HRP-TMB assay (HRP = Horse Radish Peroxidase, TMB = 3,3',5,5'-tetramethylbenzidine). Identical experiments with the unmodified FhuA Δ1-159 report no kinetics and presumably no insertion into the PIB(1000)-PEG(6000)-PIB(1000) membrane. Furthermore labeling of the Lys-NH(2) groups present in the FhuA Δ1-159 Ext channel, leads to controllability of in/out flux of substrates and products from the nanocontainer. CONCLUSION: Using a simple "semi rational" approach the protein's hydrophobic transmembrane region was increased by 1 nm, leading to a predicted lower hydrophobic mismatch between the protein and polymer membrane, minimizing the insertion energy penalty. The strategy of adding amino acids to the FhuA Δ1-159 Ext hydrophobic part can be further expanded to increase the protein's hydrophobicity, promoting the efficient embedding into thicker/more hydrophobic block copolymer membranes.

Whole Exome Sequencing Confirms Molecular Diagnostics of Three Pakhtun Families With Autosomal Recessive Epidermolysis Bullosa.

Epidermolysis bullosa (EB) is a genetic skin disorder that shows heterogeneous clinical fragility. The patients develop skin blisters congenitally or in the early years of life at the dermo-epithelial junctions, including erosions, hyperkeratosis over the palms and soles. The other associated features are hypotrichosis on the scalp, absent or dystrophic nails, and dental anomalies. Molecular diagnosis through whole-exome sequencing (WES) has become one of the successful tool in clinical setups. In this study, three Pakhtun families from the Khyber Pakhtunkhwa province of Pakistan were ascertained. WES analysis of a proband in each family revealed two novel variants (COL17A1: NM_000494.4: c.4041T>G: p.Y1347* and PLEC: NM_201380.3: c.1283_1285delGCT: p.L426del) and one previously known COL17A1: NM_000494.4:c.3067C>T: p.Q1023*) variant in homozygous forms. Sanger sequencing of the identified variants confirmed that the heterozygous genotypes of the obligate carriers. The identified variants have not only increased the mutation spectrum of the COL17A1 and PLEC but also confirms their vital role in the morphogenesis of skin and its associated appendages. WES can be used as a first-line diagnostic tool in genetic testing and counselling families from Khyber Pakhtunkhwa, Pakistan.

The First Report of a Missense Variant in RFX2 Causing Non-Syndromic Tooth Agenesis in a Consanguineous Pakistani Family.

Background: The syndromic and non-syndromic congenital missing teeth phenotype is termed tooth agenesis. Since tooth agenesis is a heterogeneous disorder hence, the patients show diverse absent teeth phenotypes. Thus identifying novel genes involved in the morphogenesis of ectodermal appendages, including teeth, paves the way for establishing signaling pathways. Methods and Results: We have recruited an autosomal recessive non-syndromic tooth agenesis family with two affected members. The exome sequencing technology identified a novel missense sequence variant c.1421T > C; p.(Ile474Thr) in a regulatory factor X (RFX) family member (RFX2, OMIM: 142,765). During the data analysis eight rare variants on various chromosomal locations were identified, but the co-segregation analysis using Sanger sequencing confirmed the segregation of only two variants RFX2: c.1421T > C; p.(Ile474Thr), DOHH: c.109C > G; p.(Pro37Ala) lying in a common 7.1 MB region of homozygosity on chromosome 19p13.3. Furthermore, the online protein prediction algorithms and protein modeling analysis verified the RFX2 variant as a damaging genetic alteration and ACMG pathogenicity criteria classified it as likely pathogenic. On the other hand, the DOHH variant showed benign outcomes. Conclusion: RFX2 regulates the Hedgehog and fibroblast growth factor signaling pathways, which are involved in the epithelial and mesenchymal interactions during tooth development. Prior animal model studies have confirmed the expression of rfx2 at a developmental stage governing mouth formation. Moreover, its regulatory role and close association with ciliary and non-ciliary genes causing various dental malformations makes it a potential candidate gene for tooth agenesis phenotype. Further studies will contribute to exploring the direct role of RFX2 in human tooth development.

Developing functionally graded PVA hydrogel using simple freeze-thaw method for artificial glenoid labrum.

Intact glenoid labrum is one of passive stabilizer for glenohumeral joint, which have various stiffness at different region. The aim of this study is to develop new artificial glenoid labrum from Polyvinyl Alcohol (PVA) hydrogel, which known as good biomaterial due to its biocompatibility and ability to tailor its modulus. PVA hydrogel was formed using freeze-thaw (FT) method and the stiffness of PVA was controlled by manipulating the concentration of PVA and number of FT cycles. Then, the gradual stiffness was formed using simple diffusion method by introducing the pre-freeze-and-thaw steps. The results showed 20% PVA with three FT cycles suit to highest stiffness of glenoid labrum while 10% PVA with three FT cycles suit to lowest stiffness of glenoid labrum. The functionally graded PVA hydrogel was then developed using the same method by diffusing two mixture (20% PVA and 10% PVA). Mechanical compression test showed, the highest modulus (0.41 MPa) found at the 20% PVA region and lowest modulus (0.1 MPa) found at 10% PVA region. While, at intermediate region, the compressive modulus was in between 20% and 10%, 0.2 MPa. The existence of gradual stiffness was further prove by checking crystallinity of material at each region using Differential Scanning Calorimetry (DSC) and Wide Angle X-ray Diffraction (WAXD). Microstructure of material was obtained from Scanning Electron Microscopy (SEM). This functionally graded PVA hydrogel also able to reduce about 51% of stress at glenoid implant and up to 17% for micromotion at the interfaces. Existence of artificial glenoid labrum could minimize the occurrence of glenoid component loosening.

Chemical Characterization, Analgesic, Antioxidant, and Anticholinesterase Potentials of Essential Oils From Isodon rugosus Wall. ex. Benth.

Isodon rugosus Wall. ex. Benth is an important species and is used in folk medicine for different types of pains such as abdominal pain, earache, toothache, gastric, and generalized body pain. Recently, we also have reported the antinociceptive potential of chloroform fraction of I. rugosus. In this research, we have investigated the antinociceptive, antioxidant and anti-cholinesterase potentials of essential oils from I. rugosus (Ir.EO), and have determined a possible mechanism of anti-nociception. The Ir.EO was subjected to gas chromatography-mass spectroscopy analysis to find out its chemical constituents. The Ir.EO was assayed for analgesic potential following acetic acid induced writhing, formalin test and hot plate method in animal models. The antioxidant activity was conducted against DPPH and ABTS free radicals following spectroscopic analysis. The cholinesterase inhibitory assays were performed using Ellman's assay. The GC-MS analysis of Ir.EO revealed the identification of 141 compounds. Ir.EO demonstrated strong antinociceptive potential in all three in-vivo models. With the use of nalaxone, it was confirmed that the essential oil was acting on the central pathway of nociception. The Ir.EO also exhibited strong free radicals scavenging potential, exhibiting IC50 values of 338 and 118 µg/ml for DPPH and ABTS free radicals respectively. In AChE and BChE inhibitory assays, the observed IC50 values were 93.56 and 284.19 µg/ml respectively. The encouraging antinociceptive, antioxidant and anticholinesterase results revealed that Ir.EO is a rich source of bioactive compounds as obvious from the GC-MS results.

Number of pegs influence focal stress distributions and micromotion in glenoid implants: a finite element study.

The present study was conducted to compare the stability of four commercially available implants by investigating the focal stress distributions and relative micromotion using finite element analysis. Variations in the numbers of pegs between the implant designs were tested. A load of 750 N was applied at three different glenoid positions (SA: superior-anterior; SP: superior-posterior; C: central) to mimic off-center and central loadings during activities of daily living. Focal stress distributions and relative micromotion were measured using Marc Mentat software. The results demonstrated that by increasing the number of pegs from two to five, the total focal stress volumes exceeding 5 MPa, reflecting the stress critical volume (SCV) as the threshold for occurrence of cement microfractures, decreased from 8.41 to 5.21 % in the SA position and from 9.59 to 6.69 % in the SP position. However, in the C position, this change in peg number increased the SCV from 1.37 to 5.86 %. Meanwhile, micromotion appeared to remain within 19-25 µm irrespective of the number of pegs used. In conclusion, four-peg glenoid implants provide the best configuration because they had lower SCV values compared with lesser-peg implants, preserved more bone stock, and reduced PMMA cement usage compared with five-peg implants.

Frequency of hepatitis "C" in Buner, NWFP.

OBJECTIVE: To assess the frequency of hepatitis C in District Buner. DESIGN: Descriptive study. PLACE AND DURATION OF STUDY: This study was conducted on outdoor patients in Medical OPD of District Headquarter (DHQ) Hospital, Daggar from January 1998 to December 2002. PATIENTS AND METHODS: A total of 16,400 patients of age between (15-70 years), who attended the outpatients department of Medical Unit at DHQ, Daggar with non-specific symptoms of dyspepsia, heart burn, bloating, generalized body aches and pain in right hypochondrium were screened for anti-HCV anti-bodies by 3rd generation ELISA. PCR and abdominal ultrasound was also done. Standard treatment was also given. Risk factors were evaluated. Population of district was regionwise divided into sectors A-D and then into subsectors. RESULTS: Out of 16,400 patients, 751 were found positive for anti-HCV antibodies (4.57%). The mean age of the patients was 37 years. The youngest was 15 years while the oldest was 65 years. The frequency of hepatitis C was higher among the male, 409/751 (54.46%) as compared to female, 342/751(45.53%). Hundred percent (751/751) had history of injections, 52/751 (6.92 %) had major surgery, 8/751 (1.06%) had blood transfusion, 73/751 (9.72%) had dental procedure, 3/751 (0.39%) had tattooing and 332/751 (44.20%) had shaving by community barbers. The highest prevalence of hepatitis C was found in sector A1(9.7%) and the lowest in sector D3(0.5%). CONCLUSION: Hepatitis C is a common health problem in District Buner, and needs proper attention to alleviate the suffering of the people. It is essential to assess the magnitude of the problem, which will help us in understanding the dynamic of its transmission for control and prevention.

Enhanced EGFP fluorescence emission in presence of PEG aqueous solutions and PIB1000-PEG6000-PIB1000 copolymer vesicles.

An EGFP construct interacting with the PIB1000-PEG6000-PIB1000 vesicles surface reported a ~2-fold fluorescence emission enhancement. Because of the constructs nature with the amphiphilic peptide inserted into the PIB core, EGFP is expected to experience a "pure" PEG environment. To unravel this phenomenon PEG/water solutions at different molecular weights and concentrations were used. Already at ~1:10 protein/PEG molar ratio the increase in fluorescence emission is observed reaching a plateau correlating with the PEG molecular weight. Parallel experiments in presence of glycerol aqueous solutions did show a slight fluorescence enhancement however starting at much higher concentrations. Molecular dynamics simulations of EGFP in neat water, glycerol, and PEG aqueous solutions were performed showing that PEG molecules tend to "wrap" the protein creating a microenvironment where the local PEG concentration is higher compared to its bulk concentration. Because the fluorescent emission can be perturbed by the refractive index surrounding the protein, the clustering of PEG molecules induces an enhanced fluorescence emission already at extremely low concentrations. These findings can be important when related to the use of EGFP as reported in molecular biology experiments.

Pott puffy tumor: a rare complication of sinusitis.

Pott puffy tumor, a rare complication of sinusitis in adults, appears as a localized swelling over the frontal region or forehead. This swelling is due to a subperiosteal abscess resulting from osteomyelitis of the underlying frontal bone. This condition may be associated with an epidural purulent collection, subdural empyema, and intracerebral abscess. This entity is rare--even forgotten--since the advent of modern powerful antibiotics. A 27-year-old male was referred to ENT OPD with forehead swelling, fever and headache. The patient had mooth doughy swelling of the foreheadwith surrounding cellulitis and swelling spreading to the orbits as periorbital odema. On examination, patient had very poor oral hygiene with severe dental caries. CT with contrast conformed pansinusitis with erosion of frontal bone and abscess. An early recognition of this forgotten complication of sinusitis will help avoid severe neurological sequelae and increased morbidity and mortality.