Performance evaluation of gravity-driven bioreactor (GDB) for simultaneous treatment of black liquor and domestic wastewater.

A lab-scale gravity-driven bioreactor (GDB) was designed and constructed to evaluate the simultaneous treatment of black liquor and domestic wastewater. The GDB was operated with a mixture of black liquor and domestic wastewater at a ratio of 1:1 and maintained at an average organic loading rate of 1235 mg-COD/L-Day. The wastewater was fed to the primary sedimentation tank at a flow rate of approximately 12 mL/min and subsequently passed through serially connected anaerobic and aerobic chambers with the same flow rate. Each wastewater sample was allowed to undergo a hydraulic retention time of approximately 72 h, ensuring effective treatment. The GDB was actively operated for nine samples (W1-W9) at a weekly frequency. The entire process was conducted within the workstation's ambient temperature range of 30-35 °C to sustain microbial activity and treatment efficiency in an open environment. The performance of the GDB was evaluated in terms of various pollution indicators, including COD, BOD5, lignin removal, TDS, TSS, EC, PO43-, SO42-, microbial load (CFU/mL and MPN index), total nitrogen, and color reduction. The results showed that the GDB achieved promising treatment efficiencies: 84.5% for COD, 71.80% for BOD5, 82.8% for TDS, 100% for TSS, 74.71% for E.C., 67.25% for PO43-, 81% for SO42-, and 69.36% for TN. Additionally, about 80% reduction in lignin content and 57% color reduction were observed after the treatment. The GDB substantially reduced microbial load in CFU/mL (77.98%) and MPN (90%). This study marks the first to report on wastewater treatment from two different sources (black liquor and domestic wastewater) using a simple GDB design. Furthermore, it highlights the GDB's potential as a cost-effective, environmentally friendly, and efficient solution for wastewater treatment, with no need for supplementary chemical or physical agents and zero operational costs.

A loss of function variant in AGPAT3 underlies intellectual disability and retinitis pigmentosa (IDRP) syndrome.

Intellectual disability (ID) and retinal dystrophy (RD) are the frequently found features of multiple syndromes involving additional systemic manifestations. Here, we studied a family with four members presenting severe ID and retinitis pigmentosa (RP). Using genome wide genotyping and exome sequencing, we identified a nonsense variant c.747 C > A (p.Tyr249Ter) in exon 7 of AGPAT3 which co-segregates with the disease phenotype. Western blot analysis of overexpressed WT and mutant AGPAT3 in HEK293T cells showed the absence of AGPAT3, suggesting instability of the truncated protein. Knockdown of Agpat3 in the embryonic mouse brain caused marked deficits in neuronal migration, strongly suggesting that reduced expression of AGPAT3 affects neuronal function. Altogether, our data indicates that AGPAT3 activity is essential for neuronal functioning and loss of its activity probably causes intellectual disability and retinitis pigmentosa (IDRP) syndrome.

Characterization of Vps13b-mutant mice reveals neuroanatomical and behavioral phenotypes with females less affected.

The vacuolar protein sorting-associated protein 13B (VPS13B) is a large and highly conserved protein. Disruption of VPS13B causes the autosomal recessive Cohen syndrome, a rare disorder characterized by microcephaly and intellectual disability among other features, including developmental delay, hypotonia, and friendly-personality. However, the underlying mechanisms by which VPS13B disruption leads to brain dysfunction still remain unexplained. To gain insights into the neuropathogenesis of Cohen syndrome, we systematically characterized brain changes in Vps13b-mutant mice and compared murine findings to 235 previously published and 17 new patients diagnosed with VPS13B-related Cohen syndrome. We showed that Vps13b is differentially expressed across brain regions with the highest expression in the cerebellum, the hippocampus and the cortex with postnatal peak. Half of the Vps13b-/- mice die during the first week of life. The remaining mice have a normal lifespan and display the core phenotypes of the human disease, including microcephaly, growth delay, hypotonia, altered memory, and enhanced sociability. Systematic 2D and 3D brain histo-morphological analyses reveal specific structural changes in the brain starting after birth. The dentate gyrus is the brain region with the most prominent reduction in size, while the motor cortex is specifically thinner in layer VI. The fornix, the fasciculus retroflexus, and the cingulate cortex remain unaffected. Interestingly, these neuroanatomical changes implicate an increase of neuronal death during infantile stages with no progression in adulthood suggesting that VPS13B promotes neuronal survival early in life. Importantly, whilst both sexes were affected, some neuroanatomical and behavioral phenotypes were less pronounced or even absent in females. We evaluate sex differences in Cohen patients and conclude that females are less affected both in mice and patients. Our findings provide new insights about the neurobiology of VPS13B and highlight previously unreported brain phenotypes while defining Cohen syndrome as a likely new entity of non-progressive infantile neurodegeneration.

Combined Single Gene Testing and Genome Sequencing as an Effective Diagnostic Approach for Anophthalmia and Microphthalmia Patients.

Anophthalmia and microphthalmia (A/M) are among the most severe congenital developmental eye disorders. Despite the advancements in genome screening technologies, more than half of A/M patients do not receive a molecular diagnosis. We included seven consanguineous families affected with A/M from Pakistani cohort and an unknown molecular basis. Single gene testing of FOXE3 was performed, followed by genome sequencing for unsolved probands in order to establish a genetic diagnosis for these families. All seven families were provided with a genetic diagnosis. The identified variants were all homozygous, classified as (likely) pathogenic and present in an A/M-associated gene. Targeted FOXE3 sequencing revealed two previously reported pathogenic FOXE3 variants in four families. In the remaining families, genome sequencing revealed a known pathogenic PXDN variant, a novel 13bp deletion in VSX2, and one novel deep intronic splice variant in PXDN. An in vitro splice assay was performed for the PXDN splice variant which revealed a severe splicing defect. Our study confirmed the utility of genome sequencing as a diagnostic tool for A/M-affected individuals. Furthermore, the identification of a novel deep intronic pathogenic variant in PXDN highlights the role of non-coding variants in A/M-disorders and the value of genome sequencing for the identification of this type of variants.

Variants in FREM1 and trisomy 18 identified in a neonatal progeria patient.

BACKGROUND: Neonatal progeroid disorders are rare disorders with clinical features including low body mass index, proptosis, aged and dysmorphic facial features at the time of birth, prominent veins, sparse scalp hairs, and severe growth retardation. Very few cases have been identified with an unknown genetic cause. Here, we report clinical and genetic findings of a proband with hallmark features of neonatal progeria. METHODS: Microarray comparative genomic hybridization, whole exome sequencing (WES) and Sanger sequencing were performed using standard methods. RESULTS: Array combined genome hybridization data revealed trisomy 18 in the proband (II-1), and WES data identified novel compound heterozygous variants (c.247 C > T; p.H83Y and c.14769868InsA) in the FREM1 gene. CONCLUSION: We report a novel complex case of neonatal progeria with atrial septal defects, trisomy 18 without typical features of Edward syndrome. The phenotype of the patient was more consistent with neonatal progeria, thus we speculate it to be caused by the FREM1 variants.

Biallelic variants in NSUN6 cause an autosomal recessive neurodevelopmental disorder.

PURPOSE: 5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive. METHODS: We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene. RESULTS: We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment. CONCLUSION: Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition.

Overlapping neurological phenotypes in two extended consanguineous families with novel variants in the CNTNAP1 and ADGRG1 genes.

BACKGROUND: Population diversity is important and rare disease isolates can frequently reveal novel homozygous or biallelic mutations that lead to expanded clinical heterogeneity, with diverse clinical presentations. METHODS: The present study describes two consanguineous families with a total of seven affected individuals suffering from a clinically similar severe syndromic neurological disorder, with abnormal development and central nervous system (CNS) and peripheral nervous system (PNS) abnormalities. Whole exome sequencing (WES) and Sanger sequencing followed by 3D protein modeling was performed to identify the disease-causing gene. RNA was extracted from the fresh blood of both families affected and healthy individuals. RESULTS: The families were clinically assessed in the field in different regions of Khyber Pakhtunkhwa. Magnetic resonance imagining was obtained in the probands and blood was collected for DNA extraction and WES was performed. Sanger sequencing confirmed a homozygous, likely pathogenic mutation (GRCh38: chr17:42684199G>C; (NM_003632.3): c.333G>C);(NP_003623.1): p.(Trp111Cys) in the CNTNAP1 gene in family A, previously associated with Congenital Hypo myelinating Neuropathy 3 (CHN3; OMIM # 618186) and a novel nonsense variant in family B, (GRCh38: chr16: 57654086C>T; NC_000016.10 (NM_001370440.1): c.721C>T); (NP_001357369.1): p.(Gln241Ter) in the ADGRG1 gene previously associated with bilateral frontoparietal polymicrogyria (OMIM # 606854); both families have extended CNS and PNS clinical manifestations. In addition, 3D protein modeling was performed for the missense variant, p.(Trp111Cys), identified in the CNTNAP1, suggesting extensive secondary structure changes that might lead to improper function or downstream signaling. No RNA expression was observed in both families affected and healthy individuals hence showing that these genes are not expressed in blood. CONCLUSIONS: In the present study, two novel biallelic variants in the CNTNAP1 and ADGRG1 genes in two different consanguineous families with a clinical overlap in the phenotype were identified. Thus, the clinical and mutation spectrum is expanded to provide further evidence that CNTNAP1 and ADGRG1 are very important for widespread neurological development.

A Novel Intronic Deletion in PDE6B Causes Autosomal Recessive Retinitis Pigmentosa by Interfering with RNA Splicing.

INTRODUCTION: Retinitis pigmentosa (RP) is a rare degenerative retinal disease caused by mutations in approximately seventy genes. Currently, despite the availability of large-scale DNA sequencing technologies, ∼30-40% of patients still cannot be diagnosed at the molecular level. In this study, we investigated a novel intronic deletion of PDE6B, encoding the beta subunit of phosphodiesterase 6 in association with recessive RP. METHODS: Three unrelated consanguineous families were recruited from the northwestern part of Pakistan. Whole exome sequencing was performed for the proband of each family, and the data were analyzed according to an in-house computer pipeline. Relevant DNA variants in all available members of these families were assessed through Sanger sequencing. A minigene-based splicing assay was also performed. RESULTS: The clinical phenotype for all patients was compatible with rod cone degeneration, with the onset during childhood. Whole exome sequencing revealed a homozygous 18 bp intronic deletion (NM_000283.3:c.1921-20_1921-3del) in PDE6B, which co-segregated with disease in 10 affected individuals. In vitro splicing tests showed that this deletion causes aberrant RNA splicing of the gene, leading to the in-frame deletion of 6 codons and, likely, to disease. CONCLUSION: Our findings further expand the mutational spectrum of the PDE6B gene.

FOXI3 pathogenic variants cause one form of craniofacial microsomia.

Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.

Shattering and yield expression of sesame (Sesamum indicum L) genotypes influenced by paclobutrazol concentration under rainfed conditions of Pothwar.

Seed shattering is a critical challenge that significantly reduces sesame production by 50%. These shattering losses can be reduced by selecting shattering resistant genotypes or by incorporating modern agronomic management such as paclobutrazol, which can boost productivity and prevent seed shattering in sesame. Two-years of field trials were conducted to examine the effect of sesame genotypes, environment, and paclobutrazol (PBZ) concentrations. Twelve sesame genotypes were used in a four-way factorial RCBD with three replications and five PBZ concentrations (T0 = Control; T1 = 150; T2 = 300; T3 = 450; and T4 = 600 mg L- 1) under rainfed conditions of Pothwar. The findings revealed significant variations in the major effects of all examined variables (genotypes, locations, years, and PBZ levels). Sesame genotypes PI-154304 and PI-175907 had the highest plant height, number of capsule plant- 1, seed capsule- 1, 1000 seed weight, biological yield, and seed yield, while also having the lowest seed losses and shattering percentage. Regarding environments, NARC-Islamabad generated the highest plant height, number of capsule plant- 1, shattering percentage, and biological yield; however, the URF-Koont produced the highest seed yield with the lowest shattering percentage. Additionally, plant height, capsules plant- 1, and biological yield were higher in 2021, while seed capsule- 1, 1000 seed weight, seed losses, shattering percentage, and seed yield were higher in 2020. PBZ concentration affected all measured parameters; plant height and number of seed capsule- 1 decreased with increasing PBZ concentrations. 450 mg L- 1 PBZ concentration generated the highest biomass, number of capsules plant- 1, and seed yield. At the same time, PBZ concentration 600 mg L- 1 generated the smallest plant, the lowest seed capsules- 1, the greatest thousand seed weight, and the lowest shattering percentage. The study concluded that paclobutrazol could dramatically reduce shattering percentage and shattering losses while increasing economic returns through better productivity. Based on the findings, the genotypes PI-154304 and PI-175907 with paclobutrazol level 450 mgL- 1 may be suggested for cultivation in Pothwar farming community under rainfed conditions, as they showed promising shattering resistance as well as enhanced growth and yield.

A Biallelic Truncating Variant in the TPR Domain of GEMIN5 Associated with Intellectual Disability and Cerebral Atrophy.

GEMIN5 is a multifunctional RNA-binding protein required for the assembly of survival motor neurons. Several bi-allelic truncating and missense variants in this gene are reported to cause a neurodevelopmental disorder characterized by cerebellar atrophy, intellectual disability (ID), and motor dysfunction. Whole exome sequencing of a Pakistani consanguineous family with three brothers affected by ID, cerebral atrophy, mobility, and speech impairment revealed a novel homozygous 3bp-deletion NM_015465.5:c.3162_3164del that leads to the loss of NM_015465.5 (NP_056280.2):p. (Asp1054_Ala1055delinsGlu) amino acid in one of the α-helixes of the tetratricopeptide repeats of GEMIN5. In silico 3D representations of the GEMIN5 dimerization domain show that this variant likely affects the orientation of the downstream sidechains out of the helix axis, which would affect the packing with neighboring helices. The phenotype of all affected siblings overlaps well with previously reported patients, suggesting that NM_015465.5: c.3162_3164del (NP_056280.2):p. (Asp1054_Ala1055delinsGlu) is a novel GEMIN5 pathogenic variant. Overall, our data expands the molecular and clinical phenotype of the recently described neurodevelopmental disorder with cerebellar atrophy and motor dysfunction (NEDCAM) syndrome.

Live Birth of a Healthy Child in a Couple with Identical mtDNA Carrying a Pathogenic c.471_477delTTTAAAAinsG Variant in the MOCS2 Gene.

Molybdenum cofactor deficiency type B (MOCODB; #252160) is an autosomal recessive metabolic disorder that has only been described in 37 affected patients. In this report, we describe the presence of an in-frame homozygous variant (c.471_477delTTTAAAAinsG) in the MOCS2 gene in an affected child, diagnosed with Ohtahara syndrome according to the clinical manifestations. The analysis of the three-dimensional structure of the protein and the amino acid substitutions suggested the pathogenicity of this mutation. To prevent transmitting this mutation to the next generation, we used preimplantation genetic testing for the monogenic disorders (PGT-M) protocol to select MOCS2 gene mutant-free embryos for transfer in an in vitro fertilization (IVF) program. As a result, a healthy child was born. Interestingly, both parents of the proband shared an identical mitochondrial (mt) DNA control region, assuming their close relationship and thus suggesting that both copies of the nuclear rare variant c.471_477delTTTAAAAinsG may have been transmitted from the same female ancestor. Our estimation of the a priori probability of meeting individuals with the same mtDNA haplotype confirms the assumption of a possible distant maternal relationship among the proband's direct relatives.

Delineating the Spectrum of Genetic Variants Associated with Bardet-Biedl Syndrome in Consanguineous Pakistani Pedigrees.

This study aimed to find the molecular basis of Bardet-Biedl syndrome (BBS) in Pakistani consanguineous families. A total of 12 affected families were enrolled. Clinical investigations were performed to access the BBS-associated phenotypes. Whole exome sequencing was conducted on one affected individual from each family. The computational functional analysis predicted the variants' pathogenic effects and modeled the mutated proteins. Whole-exome sequencing revealed 9 pathogenic variants in six genes associated with BBS in 12 families. The BBS6/MKS was the most common BBS causative gene identified in five families (5/12, 41.6%), with one novel (c.1226G>A, p.Gly409Glu) and two reported variants. c.774G>A, Thr259LeuTer21 was the most frequent BBS6/MMKS allele in three families 3/5 (60%). Two variants, c.223C>T, p.Arg75Ter and a novel, c. 252delA, p.Lys85STer39 were detected in the BBS9 gene. A novel 8bp deletion c.387_394delAAATAAAA, p. Asn130GlyfsTer3 was found in BBS3 gene. Three known variants were detected in the BBS1, BBS2, and BBS7 genes. Identification of novel likely pathogenic variants in three genes reaffirms the allelic and genetic heterogeneity of BBS in Pakistani patients. The clinical differences among patients carrying the same pathogenic variant may be due to other factors influencing the phenotype, including variants in other modifier genes.

Colletotrichum acutatum: Causal Agent of Olive Anthracnose Isolation, Characterization, and Fungicide Susceptibility Screening in Punjab, Pakistan.

Anthracnose of olive fruit caused by Colletotrichum acutatum was a severe epidemic disease in Pakistan that occurred in September 2020. The estimated disease incident was recorded as 59%. Anthracnose causes a significant reduction in yield and quality traits. Anthracnose has been found in several orchards. Agricultural practices, environmental factors, and disease aggressiveness vary between orchards. Therefore, we looked at spore size, cultural traits, morphological variation, growth pattern, and pathogenicity of different strains of C. acutatum from various orchards. Molecular and phylogenetic analysis confirmed the isolated strains as C. acutatum. In all, 15 C. acutatum isolates from olive orchards were tested for susceptibility to four commercial fungicides (P < 0.001). The examined isolates' in vitro fungicide sensitivity varied with fungicide concentration. The concentration at which conidial germination was hindered by 50% compared with the control values was observed for difenoconazole, tebuconazole, carbendazim, and cyprodinil, ranging from 0.12 to 2.69 g ml-1. Based on the findings of the fungal growth inhibition studies, carbendazim has been found to be the only fungicide that effectively reduces (P < 0.001) anthracnose caused by C. acutatum strains. Additionally, results revealed that preharvest site treatments of different fungicides greatly decreased anthracnose infections on olive fruit (70 to 90%), and postharvest site applications significantly reduced disease prevalence and severity (75 to 95%). The fungicide carbendazim significantly decreased pre- and postharvest anthracnose infection on olive cultivars. This study suggests that the latter compound might be used to control olive anthracnose in Pakistan while lowering environmental impact and fungicide resistance.

Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype.

Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

Enhancement of Magnetic and Dielectric Properties of Ni0.25Cu0.25Zn0.50Fe2O4 Magnetic Nanoparticles through Non-Thermal Microwave Plasma Treatment for High-Frequency and Energy Storage Applications.

Spinel ferrites are widely investigated for their widespread applications in high-frequency and energy storage devices. This work focuses on enhancing the magnetic and dielectric properties of Ni0.25Cu0.25Zn0.50 ferrite series through non-thermal microwave plasma exposure under low-pressure conditions. A series of Ni0.25Cu0.25Zn0.50 ferrites was produced using a facile sol-gel auto-ignition approach. The post-synthesis plasma treatment was given in a low-pressure chamber by sustaining oxygen plasma with a microwave source. The structural formation of control and plasma-modified ferrites was investigated through X-ray diffraction analysis, which confirmed the formation of the fcc cubical structure of all samples. The plasma treatment did not affect crystallize size but significantly altered the surface porosity. The surface porosity increased after plasma treatment and average crystallite size was measured as about ~49.13 nm. Morphological studies confirmed changes in surface morphology and reduction in particle size on plasma exposure. The saturation magnetization of plasma-exposed ferrites was roughly 65% higher than the control. The saturation magnetization, remnant magnetization, and coercivity of plasma-exposed ferrites were calculated as 74.46 emu/g, 26.35 emu/g, and 1040 Oe, respectively. Dielectric characteristics revealed a better response of plasma-exposed ferrites to electromagnetic waves than control. These findings suggest that the plasma-exposed ferrites are good candidates for constructing high-frequency devices.

KIF1A novel frameshift variant p.(Ser887Profs*64) exhibits clinical heterogeneity in a Pakistani family with hereditary sensory and autonomic neuropathy type IIC.

Background: Hereditary sensory and autonomic neuropathies (HSANs) are rare heterogeneous group of neurological disorders caused by peripheral nerve deterioration. The HSANs sub-clinical classes have clinical and genetic overlap which often lead to misdiagnosis. In the present study a Pakistani family with five affected members suffering from severe neuropathy were genetically analyzed to identify the disease causative element in the family.Methods: Genome wide high-density single nucleotide polymorphism (SNP) microarray analysis was carried out followed by whole exome sequencing of the affected proband and another affected sibling. Shared homozygous regions in all severely affected members were identified through homozygosity mapping approach.Results: The largest homozygous region of 14.1 Mb shared by the five severely affected members of the family was identified on chromosome 2. Subsequent exome sequencing identified a novel single nucleotide deletion c.2658del; p.(Ser887Profs*64) in KIF1A. Segregation analysis revealed that this mutation was homozygous in all five affected individuals of the family with severe clinical manifestation, while members of the family that were heterozygous carriers shared abnormal skin features (scaly skin) only with the homozygous affected members.Conclusions: A novel frameshift mutation p.(Ser887Profs*64) in KIF1A is the potential cause of severe HSANIIC in a Pakistani family along with incomplete penetrance in mutation carriers. We demonstrate that using a combination of different techniques not only strengthens the gene finding approach but also helps in proper sub-clinical characterization along with identification of mutated alleles exhibiting incomplete penetrance leading to intrafamilial clinical variability in HSAN group of inherited diseases.

Rheological, Aging, and Microstructural Properties of Polycarbonate and Polytetrafluoroethylene Modified Bitumen.

Deterioration of asphalt pavements due to massive load of vehicles and climatic variation has demanded the use of pavements construction material with an excellent resilience characteristic, resistance to permanent deformation, and most importantly, a much longer service lifespan. The main structural distresses in pavement construction are permanent deformation at high temperatures and fatigue cracking under repetitive traffic loadings. To comprehensively investigate the performance of bitumen penetration grade (PG) 70 against rutting, fatigue, and high temperature cracking in hot mix asphalt (HMA) pavements, polycarbonate (PC) and polytetrafluoroethylene (PTFE) were used. The investigation of the internal structure, rheological, and physical properties of base and modified bitumen (MB) mixes with different percentages of modifiers (0%, 2.5%, and 5%) by weight were performed via scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR) analysis, X-ray diffraction (XRD) pattern analysis, rolling thin-film oven test (RTFOT), pressurized aging vessel (PAV), dynamic shear rheometer (DSR), rotational viscosity (RV), and bending beam rheometer (BBR). The results of the RV test indicate that modification of neat bitumen with polycarbonate and polytetrafluoroethylene increased the viscosity for polycarbonate-modified bitumen (PCMB), polytetrafluoroethylene-modified bitumen (PTFEMB), and for a blend of PCMB-PTFEMB by 44%, 50%, and 55.75% at 135 °C and 111.10%, 127.80%, and 138.88% at 165 °C, accordingly. BBR test results revealed that modifiers increased the rigidity of neat bitumen by 74.8%, 75.8%, and 74.5% at -16 °C, -22 °C, and -28 °C, respectively.

Emergence of hypermucoviscous colistin-resistant high-risk convergent Klebsiella pneumoniae ST-2096 clone from Pakistan.

Klebsiella pneumoniae convergent clones are considered a threat to healthcare settings. Here we report a comprehensive genomic profiling of an emerging colistin-resistant K. pneumoniae ST-2096 convergent clone from Pakistan. Methods: Whole-genome sequencing was performed and raw reads were assembled antimicrobial resistance and virulence genes were predicted using various online tools. Results & conclusion: The phenotypically multidrug-resistant (MDR) and hypermucoviscous (hv) colistin-resistant K. pneumoniae (hvCRKP-10718), which, intriguingly, possessed a wide range of antimicrobial resistance (blaTEM-1A, blaOXA-1, blaOXA-232, blaCTX-M-15, blaSHV-106, oqxA, oqxB, aac(6')-Ib-cr, aadA2, aac(6')-Ib-cr, armA, tetD, mphE, msrE, fosA, dfrA1, dfrA12, dfrA14, catB3, sul1) and virulence determinants (RmpA/RmpA2, yersiniabactin [ybt], aerobactin [iuc/iut], enterobactin). Furthermore, the acquisition of various mobile genetic elements (MDR/virulent plasmids, type II integron gene cassette, insertional sequences, transposases) and associated hv capsular type made this MDR/hv isolate a convergent clone belonging to a high-risk lineage (ST-2096). Based on core-genome multilocus sequence typing and single-nucleotide polymorphism analysis, this isolate showed ≥99% nucleotide identity with MDR K. pneumoniae isolates from India, depicting its evolutionary background. This study provides a comprehensive genomic profiling of this high-risk convergent K. pneumoniae ST-2096 clone from Pakistan. Comparative genomics of MDR/hv colistin-resistant K. pneumoniae isolates with other MDR convergent strains from the Indian subcontinent indicated the emergence of this evolving superbug.