The transcription factor Pou4f3 is essential for the survival of postnatal and adult mouse cochlear hair cells and normal hearing.

Introduction: Hair cells (HCs) of the cochlea are responsible for sound transduction and hearing perception in mammals. Genetic mutations in the transcription factor Pou4f3 cause non-syndromic autosomal dominant hearing loss in humans (DFNA15) which varies in the age of onset depending on the individual mutation. Mouse models with germline deletion or mutations in Pou4f3 have previously demonstrated its critical role in the maturation and survival of cochlear HCs during embryonic development. However, the role of Pou4f3 in auditory function and in the survival or maintenance of cochlear HCs after birth and during adulthood has not been studied. Methods: Therefore, using the inducible CreER-loxP system, we deleted Pou4f3 from mouse cochlear HCs at different postnatal ages, relevant to specific stages of HC maturation and hearing function. Results and discussion: Elevated auditory brainstem response thresholds and significant HC loss were detected in mice with Pou4f3 deletion compared to their control littermates, regardless of the age when Pou4f3 was deleted. However, HC loss occurred more rapidly when Pou4f3 was deleted from immature HCs. Additionally, HC loss caused by Pou4f3 deletion did not affect the number of cochlear supporting cells, but caused a delayed loss of spiral ganglion neurons at 4 months after the deletion. In conclusion, Pou4f3 is necessary for the survival of cochlear HCs and normal hearing at all postnatal ages regardless of their maturation state. Our data also suggest that Pou4f3 indirectly regulates the survival of spiral ganglion neurons.

Subclinical diagnosis of cisplatin-induced ototoxicity with biomarkers.

A mouse model with cisplatin-induced ototoxicity was used in addition to human samples from the ITMAT Biobank at the University of Pennsylvania. Mouse auditory brainstem responses (ABR), inner ear histology, perilymph cisplatin sampling, and measurement of serum prestin via ELISA were performed. Human serum prestin level was measured via ELISA in patients with otological issues after cisplatin treatment and compared to matched controls. Serum prestin was significantly elevated before ABR threshold shifts in mice exposed to cisplatin compared to control mice. Prestin concentration also correlated with the severity of hearing threshold shifts in mice. After an extended rest post-cisplatin treatment, prestin returned to baseline levels in mice and humans. Prestin was significantly elevated in the serum before the onset of objective hearing loss and correlated with the severity of hearing damage indicating that prestin may function as an effective biomarker of cisplatin-induced ototoxicity. Human serum prestin levels responded similarly to mice > 3 weeks from ototoxic exposure with decreased levels of prestin in the serum.

Comparative Analysis of Waste Materials for Their Potential Utilization in Green Concrete Applications.

The utilization of solid waste in useful product is becoming a great deal of worth for individuals, organizations, and countries themselves. The powder of waste glass and silica fumes are also considered major waste materials across the globe. In this paper, the physico-chemical, thermal, and morphological properties of both waste powders are investigated in order to determine their suitability for use as a partial replacement for cement in basic concrete. They are suitable for use in concrete due to their pozzolanic and other basic properties. Extensive testing, in terms of the compressive strength test, the slump test, and the flexural strength test, has been carried out to study the replacement of cement in the range of 5-15% by waste glass powder for curing ages of 7 and 28 days. The FTIR analyses of both materials are studied for determining the effect of characteristics of chemical bonding and intense bands with bending vibrations of O-Si-O bonds. Experimental results indicate towards the potential utilization of wastes in concrete in terms of green concrete.

Microstructural and in-vitro characteristics of functional calcium silicate topcoat on hydroxyapatite coating for bio-implant applications.

The delayed tissue-implant interactions in metallic implants coated with hydroxyapatite (HA) paved the way for the development of alternative bioactive coatings. In this study, bi-layered functional gradient (HA-CS) coating was formulated by the atmospheric plasma spray (APS) process on Ti6Al4V alloy. The HA layer was applied at the metal interface to ensure long-term stability, while the calcium silicate (CS) outer layer was applied to achieve fast tissue-implant interactions. Moreover, single-layered HA and CS coating were also formulated for comparative analysis. The phase compositions, coating microstructure, chemical properties, microhardness, porosity, surface roughness, and in-vitro bioactivity were investigated. The CS top layer showed high porosity and surface roughness with respect to the inner HA layer, which constitutes an optimum microstructure to promote bioactivity. The microhardness of the outer CS layer of HA-CS was 520.3 ± 80.8 HV, while the corresponding value for the inner HA layer was 291.7 ± 45.7 HV. HA-CS and CS coatings demonstrated higher in-vitro bioactivity compared to HA coating. On the contrary, HA coating (3.76 mpy) displayed better corrosion resistance than the HA-CS (4.17 mpy) and CS coatings (4.34 mpy). The in-vitro results indicated that the HA-CS coating could promote the healthy development of osteoblast-like MG-63.

Proposed Data-Driven Approach for Occupational Risk Management of Aircrew Fatigue.

BACKGROUND: Fatigue is pervasive, under-reported, and potentially deadly where flight operations are concerned. The aviation industry appears to lack a standardized, practical, and easily replicable protocol for fatigue risk assessment which can be consistently applied across operators. AIM: Our paper sought to present a framework, supported by real-world data with subjective and objective parameters, to monitor aircrew fatigue and performance, and to determine the safe crew configuration for commercial airline operations. METHODS: Our protocol identified risk factors for fatigue-induced performance degradation as triggers for fatigue risk and performance assessment. Using both subjective and objective measurements of sleep, fatigue, and performance in the form of instruments such as the Karolinska Sleepiness Scale, Samn-Perelli Crew Status Check, Psychomotor Vigilance Task, sleep logs, and a wearable actigraph for sleep log correlation and sleep duration and quality charting, a workflow flagging fatigue-prone flight operations for risk mitigation was developed and trialed. RESULTS: In an operational study aimed at occupational assessment of fatigue and performance in airline pilots on a three-men crew versus a four-men crew for a long-haul flight, we affirmed the technical feasibility of our proposed framework and approach, the validity of the battery of assessment instruments, and the meaningful interpretation of fatigue and work performance indicators to enable the formulation of safe work recommendations. CONCLUSION: A standardized occupational assessment protocol like ours is useful to achieve consistency and objectivity in the occupational assessment of fatigue and work performance.

Ripretinib and MEK Inhibitors Synergize to Induce Apoptosis in Preclinical Models of GIST and Systemic Mastocytosis.

The majority of gastrointestinal stromal tumors (GIST) harbor constitutively activating mutations in KIT tyrosine kinase. Imatinib, sunitinib, and regorafenib are available as first-, second-, and third-line targeted therapies, respectively, for metastatic or unresectable KIT-driven GIST. Treatment of patients with GIST with KIT kinase inhibitors generally leads to a partial response or stable disease but most patients eventually progress by developing secondary resistance mutations in KIT. Tumor heterogeneity for secondary resistant KIT mutations within the same patient adds further complexity to GIST treatment. Several other mechanisms converge and reactivate the MAPK pathway upon KIT/PDGFRA-targeted inhibition, generating treatment adaptation and impairing cytotoxicity. To address the multiple potential pathways of drug resistance in GIST, the KIT/PDGFRA inhibitor ripretinib was combined with MEK inhibitors in cell lines and mouse models. Ripretinib potently inhibits a broad spectrum of primary and drug-resistant KIT/PDGFRA mutants and is approved by the FDA for the treatment of adult patients with advanced GIST who have received previous treatment with 3 or more kinase inhibitors, including imatinib. Here we show that ripretinib treatment in combination with MEK inhibitors is effective at inducing and enhancing the apoptotic response and preventing growth of resistant colonies in both imatinib-sensitive and -resistant GIST cell lines, even after long-term removal of drugs. The effect was also observed in systemic mastocytosis (SM) cells, wherein the primary drug-resistant KIT D816V is the driver mutation. Our results show that the combination of KIT and MEK inhibition has the potential to induce cytocidal responses in GIST and SM cells.

Intratympanic Diltiazem-Chitosan Hydrogel as an Otoprotectant Against Cisplatin-Induced Ototoxicity in a Mouse Model.

HYPOTHESIS: Local administration of the calcium-channel blocker (CCB), diltiazem, via intratympanic (IT) chitosan-glycerophosphate (CGP) hydrogel will protect against cisplatin-induced ototoxicity. BACKGROUND: Cisplatin induces calcium-mediated apoptosis of cochlear outer hair cells (OHCs). Previous work demonstrated otoprotection and reduced auditory brainstem response (ABR) threshold shifts in a cisplatin-induced ototoxicity mouse model treated with multiple doses of IT diltiazem given in solution. Here, we evaluated the role of a single dose of IT CGP-diltiazem as a novel otoprotectant against cisplatin-induced ototoxicity. METHODS: Baseline pure-tone and click-evoked ABRs were performed in control (IT CGP-saline, n = 13) and treatment (IT CGP-diltiazem 2 mg/kg, n = 9) groups of female CBA/J mice. A single dose of IT CGP hydrogel was administered just before intraperitoneal injection of cisplatin (14 mg/kg). On Day 7 posttreatment, ABRs were performed and cochleae were harvested. Hair cells were quantified using anti-myosin VIIa immunostaining and inner hair cell ribbon synapses were quantified using Ctbp2 immunostaining. RESULTS: There was a statistically significant effect of treatment on click- and tone-evoked ABRs between groups. The mean threshold shifts were significantly reduced in both click- and tone-evoked ABRs on Day 7 in IT CGP-diltiazem treated mice compared with CGP-saline control mice. There were no significant differences in OHC counting between groups, but there appears to be an otoprotection against loss of synapses in the apical turn from IT CGP-diltiazem treated mice (p < 0.05). CONCLUSIONS: This preliminary work suggests that IT CGP-diltiazem reduces ABR threshold shifts with possible mechanisms of protecting ribbon synapses in the setting of cisplatin-induced ototoxicity. More work is necessary to determine the mechanism underlying this otoprotection.

The hnRNP RALY regulates transcription and cell proliferation by modulating the expression of specific factors including the proliferation marker E2F1.

The heterogeneous nuclear ribonucleoproteins (hnRNP) form a large family of RNA-binding proteins that exert numerous functions in RNA metabolism. RALY is a member of the hnRNP family that binds poly-U-rich elements within several RNAs and regulates the expression of specific transcripts. RALY is up-regulated in different types of cancer, and its down-regulation impairs cell cycle progression. However, the RALY's role in regulating RNA levels remains elusive. Here, we show that numerous genes coding for factors involved in transcription and cell cycle regulation exhibit an altered expression in RALY-down-regulated HeLa cells, consequently causing impairments in transcription, cell proliferation, and cell cycle progression. Interestingly, by comparing the list of RALY targets with the list of genes affected by RALY down-regulation, we found an enrichment of RALY mRNA targets in the down-regulated genes upon RALY silencing. The affected genes include the E2F transcription factor family. Given its role as proliferation-promoting transcription factor, we focused on E2F1. We demonstrate that E2F1 mRNA stability and E2F1 protein levels are reduced in cells lacking RALY expression. Finally, we also show that RALY interacts with transcriptionally active chromatin in both an RNA-dependent and -independent manner and that this association is abolished in the absence of active transcription. Taken together, our results highlight the importance of RALY as an indirect regulator of transcription and cell cycle progression through the regulation of specific mRNA targets, thus strengthening the possibility of a direct gene expression regulation exerted by RALY.

An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm.

Separation of X- and Y-chromosome bearing sperm has been practiced for selection of desired sex of offspring to increase the profit in livestock industries. At present, fluorescence-activated cell sorter is the only successful method for separation of X- and Y-chromosome bearing sperm. This technology is based on the differences in DNA content between these two types of sperm and has been commercialized for bovine sperm. However, this technology still has problems in terms of high economic cost, sperm damage, and lower pregnancy rates compared to unsorted semen. Therefore, an inexpensive, convenient, and non-invasive approach for sperm sexing would be of benefit to agricultural sector. Within this perspective, immunological sperm sexing method is one of the attractive choices to separate X- and Y-chromosome bearing sperm. This article reviews the current knowledge about immunological approaches, viz., H-Y antigen, sex-specific antigens, and differentially expressed proteins for sperm sexing. Moreover, this review also highlighted the different methods for identification of X- and Y-sperm.

Molecular characterization of internal transcribed spacer 1 (ITS 1) region of different Trypanosoma evansi isolates of India.

Six Trypanosoma evansi isolates collected from ponies (PH1 and PK6), camel (CB2), donkeys (DJ3 and DH4) and cattle (CK5) from Haryana, Rajasthan, Uttar Pradesh and Gujarat states of India were used for molecular characterization of internal transcribed spacer 1 (ITS 1). The DNA was isolated from purified trypanosomes of these six isolates after propagation in mice model. ITS1-PCR of purified parasite DNA yielded an amplification product approximately 540 bp in size. Nucleotide sequence of ITS1 gene of CB2 isolate had 530 bp while CK5, DH4, DJ3, and PH1 isolates had 532 bp, whereas, PK6 isolates had 533 bp size. Blast data of the Indian isolates revealed 99 % homology with other available sequences of T. evansi. Multiple alignment of nucleotide sequence of ITS1 gene variants from Indian T. evansi isolates with selected homologous sequences from GenBank revealed that nucleotide substitution mostly occurred at the position of 101-103, 218-223, 243-244, 301-396 and 470-480. The isolates PH1, CK5, DH4 and DJ3 were found more associated with T. evansi isolates from the Philippines, Thailand, Iran, Egypt and China, whereas, PK6 and CB2 isolates were related to each other and were phylogenetically distant from rest of the Indian isolates used in this study. Based on the ITS1 rDNA sequence, the Neighbour-Joining consensus tree indicated clear evidence of existence of genetic diversity among T. evansi isolates from India.

Intra and inter species genetic variability of transferrin receptor gene regions in Trypanosoma evansi isolates of different livestock and geographical regions of India.

Trypanosoma evansi, the aetiological agent of Surra affects a wide range of livestock and wild animals in India. In the present study, we studied intra- and inter species genetic variability in the transferrin receptor encoding gene regions (ESAG6/7 gene region) of T. evansi isolates by cloning, sequencing and phylogenetic study collected from camel, cattle, donkeys and ponies from North-Western and Central India. The nucleotide sequence variation of ESAG6/7 gene region between Indian T. evansi isolates was up to 17.7% and amino acid sequence variation was up to 31%. Twenty nine clones from six T. evansi isolates from geographical regions of India were included into Clade 1, 5, 6, 7 and 9 consisting of ESAG6 variants reported among T. evansi isolates from South-east Asia and South America. The cladogram indicated a relation between the host species and the genetic variability in the hyper-variable region of ESAG6 gene. Analysis of the Indian ESAG6 variants and their respective Clade positions presented a host specific distribution indicating homogenous parasite population in their respective animal hosts.

Comparative efficacy of different in vitro cultivation media for Trypanosoma evansi isolated from different mammalian hosts inhabiting different geographical areas of India.

The present study was undertaken to establish an optimal medium for primary culture initiation and maintenance of T. evansi isolated from different mammalian hosts of diverse geographical regions of India viz. donkey/1 (Hardoi, Uttar Pradesh), donkey/2 (Junagarh, Gujarat), pony/1 (Hisar, Haryana), camel/1 (Bikaner, Rajasthan) which represented isolates 1, 2, 3 and 4, respectively. Primary cultures were initiated with all four isolates in five different in vitro cultivation media with seeding density of 1 × 10(6) trypanosomes/ml. The parasites of all four isolates could remain viable only for 48 h in medium E (Alsever's solution) and for 72 h in medium A, C and D. Parasites reached to a maximum density (2.5-3.75 × 10(6)/ml) within 24 h and thereafter, a sharp decline (0.5-0.75 × 10(6)/ml) in the next 72 h was observed in 1, 2 and 3 isolates cultured in medium B. In isolate 4, parasite counts got more than doubled in 24 h and then decreased gradually up to sixth day post initiation of cultivation which thereafter increased gradually up to 34 days and a constant parasite number of 10(5)/ml could be achieved for 90 days in medium B. During this prolonged culture the trypanosomes retained their long slender morphology and infectivity to mice.

Buffalo (Bubalus bubalis) term amniotic-membrane-derived cells exhibited mesenchymal stem cells characteristics in vitro.

Recent studies suggested that placentae amniotic membrane is a valuable source of stem cells in human as well as in livestock species. Advantages of amnion over other sources of stem cells included abundant availability, ethically non-objectionable and non-invasive source. The aim of the present study was the isolation, culture and characterization of amniotic-membrane-derived mesenchymal stem cells from term placentae collected postpartum in buffalo. We have observed that both presumptive epithelial-like and fibroblast-like cells were cultured and maintained from term amnion. These cells were shown the positive expression of pluripotency markers (OCT-4, SOX-2, NANOG, TERT), mesenchymal stem cell markers (CD29, CD44, CD105) and negative for haematopoietic marker (CD34) genes at different passages. In addition, these cells were also positive for alkaline phosphatase staining. Stem-ness potential of any stem cells is determined by their potential to differentiate into specific lineages of cell type. In the present study, we have successfully differentiated the amniotic-membrane-derived cells into adipogenic, chondrogenic and osteogenic lineages of cells in vitro. In conclusion, the results of this study demonstrate that amniotic-membrane-derived cells expressed pluripotent and mesenchymal stem cells markers and have propensity to differentiate into cells of mesenchymal lineage cell type upon directed differentiation in vitro.

Inherited and Somatic Defects in DDX41 in Myeloid Neoplasms.

Most cases of adult myeloid neoplasms are routinely assumed to be sporadic. Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases led to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing. DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

FSH-FSHR3-stem cells in ovary surface epithelium: basis for adult ovarian biology, failure, aging, and cancer.

Despite extensive research, genetic basis of premature ovarian failure (POF) and ovarian cancer still remains elusive. It is indeed paradoxical that scientists searched for mutations in FSH receptor (FSHR) expressed on granulosa cells, whereas more than 90% of cancers arise in ovary surface epithelium (OSE). Two distinct populations of stem cells including very small embryonic-like stem cells (VSELs) and ovarian stem cells (OSCs) exist in OSE, are responsible for neo-oogenesis and primordial follicle assembly in adult life, and are modulated by FSH via its alternatively spliced receptor variant FSHR3 (growth factor type 1 receptor acting via calcium signaling and the ERK/MAPK pathway). Any defect in FSH-FSHR3-stem cell interaction in OSE may affect folliculogenesis and thus result in POF. Ovarian aging is associated with a compromised microenvironment that does not support stem cell differentiation into oocytes and further folliculogenesis. FSH exerts a mitogenic effect on OSE and elevated FSH levels associated with advanced age may provide a continuous trigger for stem cells to proliferate resulting in cancer, thus supporting gonadotropin theory for ovarian cancer. Present review is an attempt to put adult ovarian biology, POF, aging, and cancer in the perspective of FSH-FSHR3-stem cell network that functions in OSE. This hypothesis is further supported by the recent understanding that: i) cancer is a stem cell disease and OSE is the niche for ovarian cancer stem cells; ii) ovarian OCT4-positive stem cells are regulated by FSH; and iii) OCT4 along with LIN28 and BMP4 are highly expressed in ovarian cancers.

Cultured buffalo umbilical cord matrix cells exhibit characteristics of multipotent mesenchymal stem cells.

Recent findings have demonstrated umbilical cord, previously considered as a biomedical waste, as a source of stem cells with promising therapeutic applications in human as well as livestock species. The present study was carried out to isolate the umbilical cord matrix cells and culture for a prolonged period, cryopreserve these cells and test their post-thaw viability, characterize these cells for expression of stem cell markers and differentiation potential in vitro. The intact umbilical cord was taken out of the amniotic sac of a fetus and then incised longitudinally to remove umbilical vessels. Wharton's jelly containing tissue was diced into small pieces and placed in tiny drops of re-calcified buffalo plasma for establishing their primary culture. Confluent primary culture was trypsinized and passaged with a split ratio of 1:2 for multiplication of cells. Cryopreservation of cells was performed at three different passages in cryopreservation medium containing 15%, 20% and 25% fetal bovine serum (FBS). A significant increase in post-thaw viability was observed in cells cryopreserved in freezing medium with higher concentration of FBS. After re-culturing, frozen-thawed cells started adhering, and spike formation occurred within 4-6 h with similar morphology to their parent representative cultures. The normal karyotype and positive expression of alkaline phosphatase and pluripotency genes OCT4, NANOG and SOX2 were observed at different passages of culture. When induced, these cells differentiated into adipogenic and osteogenic cells as confirmed by oil red O and alizarin red stains, respectively. This study indicates that buffalo umbilical cord matrix cells have stemness properties with mesenchymal lineage restricted differentiation and limited proliferation potential in vitro.

SF3B1 haploinsufficiency leads to formation of ring sideroblasts in myelodysplastic syndromes.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

Seizures in acute childhood stroke.

OBJECTIVES: To describe the risk of seizures in children with acute stroke and identify factors predicting their later risk of epilepsy. STUDY DESIGN: Data for patients >3.5 years of age at a tertiary care children's hospital with acute stroke were collected and reviewed. RESULTS: Seventy-seven patients were identified (mean age, 8.4 years); 21% had clinical seizures. An additional 10% of patients had a clinical seizure during the acute hospitalization. Status epilepticus was common in infants and patients with cortical strokes. Non-convulsive status epilepticus was captured only in patients with prolonged electroencephalograms and always within 24 hours of monitoring. Six months after their stroke, 24% of our patients had epilepsy, all of whom experienced seizures at initial presentation with stroke. CONCLUSION: In our series of pediatric patients with stroke, most of the clinical seizures occurred within the first 24 hours of presentation and did not vary in stroke subtype. Status epilepticus was common, especially in infants. Epilepsy had a high likelihood of developing in the next 6 months in children with seizures in the first 24 hours of stroke onset. Prolonged electroencephalogram monitoring was useful in detecting non-convulsive status epilepticus, but not in predicting the risk of epilepsy at 6 months.

Mutations in U4atac snRNA, a component of the minor spliceosome, in the developmental disorder MOPD I.

Small nuclear RNAs (snRNAs) are essential factors in messenger RNA splicing. By means of homozygosity mapping and deep sequencing, we show that a gene encoding U4atac snRNA, a component of the minor U12-dependent spliceosome, is mutated in individuals with microcephalic osteodysplastic primordial dwarfism type I (MOPD I), a severe developmental disorder characterized by extreme intrauterine growth retardation and multiple organ abnormalities. Functional assays showed that mutations (30G>A, 51G>A, 55G>A, and 111G>A) associated with MOPD I cause defective U12-dependent splicing. Endogenous U12-dependent but not U2-dependent introns were found to be poorly spliced in MOPD I patient fibroblast cells. The introduction of wild-type U4atac snRNA into MOPD I cells enhanced U12-dependent splicing. These results illustrate the critical role of minor intron splicing in human development.