Diversity and distribution of fish in the Qilian Mountain Basin.

The Qilian Mountain Basin, on the north-eastern edge of the Qinghai-Tibet Plateau (QTP), supports a high diversity of native and endemic fish. However, the detailed species inventory and distribution patterns concerning fish in the whole Basin remain unknown, which hinders the conservation of biodiversity and assessment of ecological health. We compiled a comprehensive species richness and distribution database of freshwater fish in the Qilian Mountain Basin, based on field investigations and exhaustive data collection from 50 rivers or lakes. Then, we elucidated a distribution pattern using clustering and ordination analyses based on a ßdissim matrix with species presence/absence data. A total of 79 freshwater fish species within eight orders, 17 families and 42 genera were recorded. The Qilian Mountain Basin could be grouped into six systems, which match the six Basins (i.e. Heihe River Basin, HHR; Qaidam Basin, QDM; Qinghai Lake Basin, QHL; Shule River Basin, SLR; Shiyang River Basin, SYR; Yellow River Basin, YR), based on the fish distribution pattern. Additionally, the spatial pattern of species distribution showed the distance decay of taxonomic similarity. Our results demonstrate that riverine connectivity resulting from historical processes plays a vital role in shaping the freshwater ichthyofauna of High Central Asia. These findings will be valuable for future systematic conservation of fish in the Qilian Mountain Basin.

Renal agenesis-related genes are associated with Herlyn-Werner-Wunderlich syndrome.

OBJECTIVE: To explore the genetic causes of Herlyn-Werner-Wunderlich syndrome (HWWS) using whole-exome sequencing. DESIGN: Retrospective genetic study. SETTING: Academic medical center. PATIENT(S): Twelve patients with HWWS. INTERVENTION(S): Whole-exome sequencing was performed for each patient. Sanger sequencing was used to confirm the potential causative genetic variants. In silico analysis and American College of Medical Genetics and Genomics guidelines were used to classify the pathogenicity of each variant. MAIN OUTCOME MEASURE(S): Rare sequence variants associated with müllerian duct development and renal agenesis were identified and included in subsequent analyses. RESULT(S): A total of 11 variants were identified in 10 of 12 patients (83.3%) and were considered to constitute a molecular genetic diagnosis of HWWS. These 11 variants were related to 9 genes: CHD1L, TRIM32, TGFBR3, WNT4, RET, FRAS1, FAT1, FOXF1, and PCSK5. All variants were heterozygous and confirmed by Sanger sequencing. The changes included one frameshift variant, one splice-site variant, and eight missense variants. All of the identified variants were absent or rare in Genome Aggregation Database East Asian populations. One of the 11 variants (9.1%) was classified as a pathogenic variant according to the American College of Medical Genetics and Genomics guidelines, and 8 of the 11 variants (72.7%) were classified as variants of uncertain significance. CONCLUSION(S): To our knowledge, this is the first report of the genetic causes of HWWS. Renal agenesis-related genes, such as CHD1L, TRIM32, RET, and WNT4, may be associated with HWWS. Identification of these variants can not only help us understand the etiology of HWWS and the relationship between reproductive tract development and urinary system development, but additionally improve the level of genetic counseling for HWWS.

Sequence variant in the CDC42BPB gene is potentially associated with Mullerian duct anomalies.

AIM: Mullerian duct anomalies (MDA) are common female genital tract malformations. Genetic and environmental factors are important causes of MDA in women. Although many genes and mutations have been found to be associated with the pathogenesis of MDA, in most cases, the genetic pathogenic factors of MDA are still unknown. METHODS: We first analyzed the three sisters using low coverage whole-genome sequencing. Then whole-exome sequencing was carried out in each patient. The identified sequence variant was confirmed by Sanger sequencing. In silico pathogenicity analysis and conservative analysis of the mutation site were also performed. Protein structural modeling was used to analyze the effect of the mutated amino acid. RESULTS: We first analyzed the three sisters with septate uterus using low coverage whole-genome sequencing, but no possible pathogenic copy number variation was found. Then whole-exome sequencing was performed on the three sisters, and a rare homozygous variant, CDC42BPB:c.2012G>A:p.R671Q, was identified. All three patients were found with this variant. Sanger sequencing validated that this variant was segregated within the family. In silico pathogenicity analysis and conservative analysis of the mutation site suggested that the variant might be damaging. Protein structural analysis suggested that R671Q might weaken the electrostatic potential of this region, which may be a significant regulation target or protein interaction surface of CDC42BPB. CONCLUSION: We demonstrated that CDC42BPB genetic variant might be potentially associated with the pathogenesis of MDA.

Extracellular matrix-derived biomaterials in engineering cell function.

Extracellular matrix (ECM) derived components are emerging sources for the engineering of biomaterials that are capable of inducing desirable cell-specific responses. This review explores the use of biomaterials derived from naturally occurring ECM proteins and their derivatives in approaches that aim to regulate cell function. Biomaterials addressed are grouped into six categories: purified single ECM proteins, combinations of purified ECM proteins, cell-derived ECM, tissue-derived ECM, diseased and modified ECM, and ECM-polymer coupled biomaterials. Purified ECM proteins serve as a material coating for enhanced cell adhesion and biocompatibility. Cell-derived and tissue-derived ECM, generated by cell isolation and decellularization technologies, can capture the native state of the ECM environment and guide cell migration and alignment patterns as well as stem cell differentiation. We focus primarily on recent advances in the fields of soft tissue, cardiac, and dermal repair, and explore the utilization of ECM proteins as biomaterials to engineer cell responses.

Whole-Exome Sequencing Identified a TBX6 Loss of Function Mutation in a Patient with Distal Vaginal Atresia.

STUDY OBJECTIVE: The purpose of this study was to determine if there are any genetic changes with whole-exome sequencing associated with distal vaginal atresia. DESIGN: This was a retrospective genetics study of 5 patients who presented with distal vaginal atresia who were recruited between 2017 and 2018. Whole-exome sequencing was performed in each subject with distal vaginal atresia. Sanger sequencing was used to confirm the potential causative genetic mutation. SETTING: Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China. PARTICIPANTS AND MAIN OUTCOME MEASURES: The main outcome measure was the rare mutations potentially associated with distal vaginal atresia in 5 patients. RESULTS: A truncating mutation c.266delC (p.P89Rfs*5) in the T-box transcription factor 6 (TBX6) gene, which is highly expressed in the human vagina, was identified in 1 patient using whole-exome sequencing. The deletion of the 16p11.2 region containing the TBX6 locus has also been reported previously to have the clinical feature of Müllerian agenesis. This mutation was paternally inherited by the patient. This truncating mutation was absent from all of the databases we checked, suggesting that the variant is rare and pathogenic. CONCLUSION: We showed, to our knowledge, for the first time, that the mutation in TBX6 might be associated with human distal vaginal atresia.

Identification of TRADD as a potential biomarker in human uterine leiomyoma through iTRAQ based proteomic profiling.

Recurrent and refractory leiomyoma of uterus is one of the most common diseases in women of reproductive age. Despite its benign nature, uterine leiomyoma has presented an extremely deleterious impact on public health. The etiology of uterine leiomyoma remains unclear and clinical management remains suboptimal, leaving radical hysterectomy the only effective approach. Delineating the molecular mechanism underlying the leiomyoma initiation and progression remains an unmet clinical need. To screen proteins that were differentially expressed in uterine leiomyoma versus normal myometrium, we examined proteomic profile by isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS). 72 proteins have been identified as differentially expressed in uterine leiomyoma, including the downregulation of TRADD (tumor necrosis factor receptor type 1-associated DEATH domain protein), which dominates the dysfunctional extrinsic apoptosis pathway and deregulated inflammatory responses. The reduction of TRADD was further validated by Western blot and immunohistochemistry in independent sample cohorts. Our data thus suggest potential biological significance of TRADD mediated inflammatory response in the development of uterine leiomyoma.

CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in patients on long-term treatment: HIV-1 p24-producing cells and suppression of anti-HIV immunity.

BACKGROUND: A major question when attempting to eradicate and treat HIV-1 infection is how to reactivate latent proviruses. Stimulating HIV-1-specific cytolytic T lymphocytes (CTL) has been shown to facilitate the elimination of the latent viral reservoir after viral reactivation. Regulatory T (Treg) cells are known to be capable of lowering both HIV-specific immunoreactions and general immune activation during HIV-1 infection. It was hypothesized that the depletion of Treg cells could increase the HIV-1-specific cytolytic T lymphocyte response and reactivate HIV-1 p24 production. METHODS: Treg cells were isolated by isolation kit according to the surface marker of Treg cells. Real-time PCR method was used to quantify HIV-1 DNA. P24 antigens in the cell culture supernatant was done by ELISA. Cells activation and HIV specific HIV-1 CD8+ T cells were analyses using a FACSCalibur flow cytometer and CELLQUEST software. RESULTS: This study included both HIV-infected patients who were antiviral treatment-naïve and patients with sustained viral responses to antiretroviral therapy (ART) for 1 or 5 years. It was found that the HIV-DNA levels in Treg cells were approximately 10-fold higher than those in non-Treg CD4+ cells and that the depletion of Treg cells could enhance the frequency of HIV-1-specific CTL and immune activation after 5 years of effective ART. CONCLUSIONS: CD4+CD25+CD127 regulatory cells play multiple roles in maintaining HIV-1 p24 production in long-term ART patients. Treg cells may be a target for eliminating the latent HIV reservoir after effective long-term ART.

[Establishing a 29 Y-STR Loci Multiplex PCR System].

OBJECTIVE: To establish a 29 Y-STR loci multiplex PCR system for investigating the genetic polymorphisms and to assess its application value in forensic science. METHODS: A multiplex PCR system was established using a five color fluorescence labeling 29 Y-STR loci (DYS456, DYS389 I , DYS437, DYS447, DYS389 11, DYS438, DYS522, DYS460, DYS458, DYS622, DYS390, DYS392, DYS448, DYS449, DYS391, Y-GA TA-H4, DYS388, DYS19, DYS385a/b, DYS527a/b, DYS393, DYS459a/b, DYS635, DYS439, DYS570 and DYS627) for multiple amplification and capillary electrophoresis. And its applicability was validated with genetic polymorphism data of 29 Y-STR of unrelated 2,000 male samples in Shandong Han population. RESULTS: A total of 1,981 different haplotypes of 2,000 individuals showed genotype diver- sity between 0.370 0 and 0.965 4. The system provided stable and accurate typing with high sensitivity of 0.05 ng. It satisfied the needs of variety of routine biological samples. CONCLUSION: The 29 Y-STR loci multiplex PCR system could be applied for actual cases and establishment of Y-STR database. In addition, it has great significance in forensic science practices and related research.

Molecular portraits of heterogeneity related to cancer stem cells in human ovarian cancer.

PURPOSE: The aim of this study was to investigate molecular portraits of heterogeneity related to cancer stem cells (CSCs) in human ovarian cancer and to access the value in diagnosis and treatment. METHODS: Sixty specimens were collected in both cytoreductive and re-cytoreductive surgeries of 20 serous papillary ovarian adenocarcinoma cases. Expression density and distribution of 3 CSC markers (CD44, CD133, and CD117) and 3 stemness proteins (Bmi1, Nestin, and Oct3/4) were analyzed by immunohistochemical staining. Pairwise comparisons were performed among their expression in primary, metastasis, and relapsing tumors. RESULTS: Some molecules presented different localization in 1 tissue, like CD133 and CD117, and all but Oct3/4 expressed differentially in different specimens of 1 case. Compared to primary or metastatic cancers, recurrent cancers show higher expression of CD133, CD117, and Bmi1, as well as higher histological grades. CONCLUSIONS: Our study indicated that there exist extratumoral and intratumoral heterogeneity in ovarian epithelial cancers related to CSCs. And this is worth further studying.

Identification of a cancer stem cell-like side population in the HeLa human cervical carcinoma cell line.

The present study aimed to identify the stem cell characteristics of side population (SP) cells sorted from the widely-used HeLa human cervical carcinoma cell line. The SP cells were sorted from the HeLa cell line using fluorescence-activating cell sorting (FACS). Stem cell characteristics of the SP cells, including proliferation, self-renewal, differentiation and the ability to form xenografts, were investigated in vitro and in vivo. The SP cells demonstrated strong tumorigenesis following in vivo transplantation into five to six-week-old female Balb/c mice. The SP cells were observed to be more resistant to chemotherapy and radiotherapy compared with non-side population (NSP) cells. A higher expression of CD133 was observed in the SP cells compared with the NSP cells following FACS. The results demonstrated that the SP cells from the HeLa human cervical carcinoma cell line exhibit stem cell characteristics in vitro and also have a strong ability to form tumors in vivo. The cell surface marker CD133 may serve as a potential molecular marker for the identification of cervical cancer stem cells (CSCs).

[Analysis of the characteristics of side population cells in the human ovarian cancer cell line OVCAR-3].

OBJECTIVE: To identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells. METHODS: SP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342. Limiting dilution transplantation assay, real-time PCR, and drug sensitivity assay were performed to compare the tumorigenic ability, differentiation ability in vivo, the mRNA expression of "stemness" marker (Oct-4, Klf4, and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2, ABCB1, and ABCC2), and response to multiple drugs (cisplatin, paclitaxel, doxorubicin, and mitoxantrone) between SP and NSP cells. RESULTS: A few of SP cells [(1.13 ± 0.39)%] which were sensitive to reserpine were identified in OVCAR-3 cells. The injection of as few as 10(2) SP cells initiated tumors in two of five mice. Tumor latency was 52 - 61 days. However, the NSP cells did not generate any tumors in mice until 10(4) NSP cells were injected (two of five mice). Tumor latency was 64 - 98 days. Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells. The SP cells regenerated both SP [(2.09 ± 0.73)%] and NSP populations in vivo with a fraction size that was comparable to the original population. The mRNA expression of "stemness" genes Oct-4, Klf4 and ABC transporters ABCG2, ABCC2 genes were elevated in SP cells compared to NSP cells, the fold changes were 1.95 ± 0.41 (P < 0.05), 4.26 ± 0.63 (P < 0.01), 3.22 ± 0.36 (P < 0.01), and 1.76 ± 0.26 (P < 0.01), respectively. The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P < 0.01), 0.521 ± 0.092 versus 0.384 ± 0.073 (P < 0.05), 0.742 ± 0.051 versus 0.526 ± 0.088 (P < 0.01), and 0.690 ± 0.096 versus 0.466 ± 0.112 (P < 0.01) when they exposed to 0.25 µg/ml cisplatin, 0.01 µmol/L paclitaxel, 0.25 µmol/L doxorubicin, and 0.05 µg/ml mitoxantrone, respectively. CONCLUSIONS: SP cells from OVCAR-3 have enhanced self-renewal, differentiation, and tumor-initiating capacity compared to NSP cells. The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells, which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells. Therefore, SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.

Ovarian cancer cells with the CD117 phenotype are highly tumorigenic and are related to chemotherapy outcome.

Cancer stem cells (CSCs) play an important role in the recurrence and drug resistance of cancer. Isolation and characterization of CSCs from ovarian cancer samples may help to provide novel diagnostic and therapeutic targets in the management of recurrent disease and drug resistance in ovarian cancer. Here, we developed a xenograft model in which cells from 14 samples of human ovarian serous adenocarcinoma tissue or ascites were implanted in immunodeficient mice to test the tumorigenic potential of different populations of ovarian cancer cells. We identified and isolated the tumorigenic cells as CD117(+)Lineage(-) from three different xenografts. As few as 10(3) cells with the CD117(+)Lineage(-) phenotype, which comprise <2% of the xenograft tumor cells, were able to regenerate tumors in a mouse model, a 100-fold increase in tumorigenic potential compared to CD117(-)Lineage(-) cells. The tumors that arose from purified CD117(+)Lineage(-) cells reproduced the original tumor heterogeneity and could be serially generated, demonstrating the ability to self-renew and to differentiate, two defining properties of stem cells. Furthermore, immunohistochemistry analysis of 25 patients with advanced ovarian serous adenocarcinoma revealed positive immunostaining for CD117 in 40% (10 of 25) of patients. CD117 expression was statistically correlated with resistance to conventional chemotherapy (P=0.027). In conclusion, our study demonstrates that human ovarian cancer cells with the CD117(+) phenotype possess the unique properties of CSCs, including self-renewal, differentiation, a high tumorigenic potential, and chemoresistance. Future studies designed to target CD117(+) cancer cells may identify more attractive and effective therapies for treatment of ovarian cancer.

The effect of pedicle screw insertion through the neurocentral cartilage on the growth of immature canine vertebra.

OBJECTIVE: To explore the influence of pedicle screw insertion through the neurocentral cartilage (NCC) on the development of vertebrae and spinal canal in an animal experiment. METHODS: Sixteen dogs were randomly assigned to three groups: in group 1, posterior muscles at the surgery site were dissected; in group 2, the pedicles were drilled through the NCC by screws; in group 3, screws were placed in the pedicles through the NCC. Vertebrae of T8, T10, T12, L2, L4 and L6 were studied with the average data of the adjacent two vertebrae serving as controls. Spiral computerized tomography (CT) was used to assess the morphologic parameters of studied vertebrae and their controls. Measurements were made by an independent radiologist on the first post-operative day and 3 months after operation. Paired Student's t-tests of studied vertebrae and their controls were performed to evaluate the effect of pedicle screw insertion. RESULTS: In group 3, 3 months after operation the area, transverse diameter and anterior-posterior diameter of the vertebral canal and length of pedicle of studied vertebrae were significantly smaller than those of control vertebrae (P < 0.05). There were no significant differences in morphologic parameters between the studied vertebrae and the control vertebrae in groups 1 and 2 (P > 0.05). CONCLUSION: (i) Pedicle screw placement has a significant impact on the growth of the canine vertebra canal, and may lead to iatrogenic spinal stenosis, but their placement has no significant effect on the vertebral bodies; and (ii) the NCC can repair itself automatically. Drilling pedicle bone through the NCC with a screw and then removing the screw has no obvious impact on the growth of vertebrae.