GPCR Signaling Measurement and Drug Profiling with an Automated Live-Cell Microscopy System.

A major limitation of time-lapse microscopy combined with fluorescent biosensors, a powerful tool for quantifying spatiotemporal dynamics of signaling in single living cells, is low-experimental throughput. To overcome this limitation, we created a highly customizable, MATLAB-based platform: flexible automated liquid-handling combined microscope (FALCOscope) that coordinates an OpenTrons liquid handler and a fluorescence microscope to automate drug treatments, fluorescence imaging, and single-cell analysis. To test the feasibility of the FALCOscope, we quantified G protein-coupled receptor (GPCR)-stimulated Protein Kinase A activity and cAMP responses to GPCR agonists and antagonists. We also characterized cAMP dynamics induced by GPR68/OGR1, a proton-sensing GPCR, in response to variable extracellular pH values. GPR68-induced cAMP responses were more transient in acidic than neutral pH values, suggesting a pH-dependence for signal attenuation. Ogerin, a GPR68 positive allosteric modulator, enhanced cAMP response most strongly at pH 7.0 and sustained cAMP response for acidic pH values, thereby demonstrating the capability of the FALCOscope to capture allosteric modulation. At a high concentration, ogerin increased cAMP signaling independent of GPR68, likely via phosphodiesterase inhibition. The FALCOscope system thus enables enhanced throughput single-cell dynamic measurements and is a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells and for drug profiling and screening.

Exploring the nursing effect of application Albizia bark on autism in children based on network pharmacology and molecular docking.

OBJECTIVE: Autism is a disorder that manifests itself in early childhood. Early diagnosis of autism may not only help the affected children themselves, but also affect family well-being and social stability. The natural drug Albizia bark has been reported to have some effect in the prevention and treatment of autism in children. Therefore, we used network pharmacology and molecular docking to explore the possible mechanism. MATERIALS AND METHODS: TCMID and BATMAN-TCM was used to retrieve the chemical constituents of Albizia bark, and then obtained the relevant targets about autism by TTD, Gene Cards and OMIM. The resulting ingredients and targets were predicted, then a protein interaction network was constructed, and finally bioinformatics analysis was performed. Finally, molecular docking was used to verify the effective ingredients and targets obtained from the screening. RESULTS: Leucaena saponin B, luteolin, 3', 4', 7-trihydroxyflavone, which may be the key compounds for the treatment of autism. BP mainly involving signal transduction, G protein coupled receptor signal pathway, protein phosphorylation. CC, mainly involving plasma membrane, integral component of plasma membrane, MF, including protein binding, adenosine triphosphate binding, protein kinase activity. Molecular docking showed that AKT1, HRAS, PIK3CA, PIK3R1 and SRC, five potential targets, had good binding ability to Leucaena saponin B. CONCLUSIONS: The natural drug Albizia bark exerts pharmacological effects in a multi-component, multi-target and multi-channel manner, including neural regulation, inflammatory response and immune regulation.

[Establishment of human colon cancer transplantation tumor model in normal immune mice].

Objective: Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity. Methods: Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue. Results: After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm(3) 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion: Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.

[Association analysis of famine exposure during early life and risk of hypertension in adulthood].

Objective: To explore the relationship between exposure to famine in early life and the risk of hypertension in adulthood. Methods: The medical data of Yichang Health Management Big Data Center from 2018 to 2019 were analyzed. A retrospective cohort study design was adopted, with hypertension as the study outcome, and different life periods exposed to the Great Famine in China were divided into groups. Multivariate logistic regression model was used to analyze the relationship between famine exposure in early life and hypertension in adulthood. At the same time, the interaction between gender and famine exposure was analyzed. Results: The age of 142 016 subjects was (60. 56±4.43). Among them, men accounted for 46.36% (65 845/142 016) and women accounted for 53.64% (76 171/142 016). There are 42 575(29.98%), 19 644(13.83%), 28 405(20.00%), 28 305(19.93%), 23 087 (19.93%) in non-famine exposure group, fetal famine exposure group, early childhood famine exposure group and late childhood famine exposure group, respectively. The prevalence of hypertension was 17.57% (24 947 cases). Multivariate logistic regression model analysis showed that after adjusting for related confounding factors, compared with non-famine exposure group, the risk of hypertension in fetal, early childhood, middle childhood and late childhood famine exposure group was higher and the OR (95%CI) values were 1.16 (1.11-1.22), 1.27 (1.21-1.33), 1.54 (1.47-1.60) and 1.84 (1.76-1.92), respectively. There was an interaction between sex and famine exposure group (P<0.001). The above association is stronger among women than among men. Conclusion: Famine exposure in early life may increase the risk of hypertension in adulthood, and the risk of women is greater than that of men.

Hsa_circ_0007534 knockdown represses the development of colorectal cancer cells through regulating miR-613/SLC25A22 axis.

The article "Hsa_circ_0007534 knockdown represses the development of colorectal cancer cells through regulating miR-613/SLC25A22 axis, by D.-Y. Ding, D. Wang, Z.-B. Shu, published in Eur Rev Med Pharmacol Sci 2020; 24 (6): 3004-3022-DOI: 10.26355/eurrev_202003_20665-PMID: 32271418" has been withdrawn from the authors stating that "the findings are not reliable. The dosage was significantly low. The results can be misleading to many readers". The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20665.

Hsa_circ_0007534 knockdown represses the development of colorectal cancer cells through regulating miR-613/SLC25A22 axis.

OBJECTIVE: Colorectal cancer (CRC) is a common tumor around the world. Circular RNAs (circRNAs) have been reported to be related to the development of CRC. However, the detailed mechanism is complicated. This study aimed to reveal the functional mechanism of circ_0007534 in CRC. PATIENTS AND METHODS: Quantitative real time polymerase chain reation (qRT-PCR) and Western blot assay were performed to analyze gene expression. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay were carried out to determine cell proliferation ability. Furthermore, cell migratory and invasive abilities were assessed by transwell assay. Glycolytic metabolism was examined via the measurements of extracellular acidification rate (ECAR), glucose consumption, and lactate production. Also, the interaction between circ_0007534 or solute carrier family 25 member 22 (SLC25A22) and miR-613 was predicted and confirmed by starBase v2.0 and the Dual-Luciferase reporter assay, respectively. Mouse xenograft was performed to investigate the effect of circ_0007534 on tumor growth in vivo. RESULTS: Circ_0007534 and SLC25A22 levels were upregulated, and miR-613 level was downregulated in CRC tissues/cells. Circ_0007534 knockdown repressed CRC cell proliferation, colony formation, migration, invasion, and glycolysis. Interestingly, Circ_0007534 targeted miR-613, and miR-613 targeted SLC25A22. Circ_0007534 exerted its function by repressing miR-613 expression, and miR-613 exerted its function via inhibiting SLC25A22 expression. Also, Circ_0007534 repressed miR-613 expression to upregulate SLC25A22 level. Circ_0007534 depletion repressed tumor growth in vivo. CONCLUSIONS: We demonstrated that circ_0007534 knockdown suppressed the growth of CRC cells by regulating miR-613/SLC25A22 axis, providing potential target for the treatment of CRC.

Proton-sensing G protein-coupled receptors: detectors of tumor acidosis and candidate drug targets.

Cells in tumor microenvironments (TMEs) use several mechanisms to sense their low pH (<7.0), including via proton-sensing G protein-coupled receptors (psGPCRs): GPR4, GPR65/TDAG8, GPR68/OGR1 and GPR132/G2A. Numerous cancers have increased expression of psGPCRs. The psGPCRs may contribute to features of the malignant phenotype via actions on specific cell-types in the TME and thereby promote tumor survival and growth. Here, we review data regarding psGPCR expression in tumors and cancer cells, impact of psGPCRs on survival in solid tumors and a bioinformatics approach to infer psGPCR expression in cell types in the TME. New tools are needed to help define contributions of psGPCRs in tumor biology and to identify potentially novel therapeutic agents for a variety of cancers.

GPCRs in pancreatic adenocarcinoma: Contributors to tumour biology and novel therapeutic targets.

Pancreatic cancer has one of the highest mortality rates (5-year survival ~9%) among cancers. Pancreatic adenocarcinoma (PAAD) is the most common (>80%) and the most lethal type of pancreatic cancer. A need exists for new approaches to treat pancreatic adenocarcinoma. GPCRs, the largest family of cell-surface receptors and drug targets, account for ~35% of approved drugs. Recent studies have revealed roles for GPCRs in PAAD cells and cells in the tumour micro-environment. This review assesses current information regarding GPCRs in PAAD by summarizing omics data for GPCRs expression in PAAD. The PAAD "GPCRome" includes GPCRs with approved agents, thereby offering potential for their repurposing/repositioning. We then reviewed the evidence for functional roles of specific GPCRs in PAAD. We also highlight gaps in understanding the contribution of GPCRs to PAAD biology and identify several GPCRs that may be novel therapeutic targets for future work in search of GPCR-targeted drugs to treat PAAD tumours.

iPSC modeling of young-onset Parkinson's disease reveals a molecular signature of disease and novel therapeutic candidates.

Young-onset Parkinson's disease (YOPD), defined by onset at <50 years, accounts for approximately 10% of all Parkinson's disease cases and, while some cases are associated with known genetic mutations, most are not. Here induced pluripotent stem cells were generated from control individuals and from patients with YOPD with no known mutations. Following differentiation into cultures containing dopamine neurons, induced pluripotent stem cells from patients with YOPD showed increased accumulation of soluble α-synuclein protein and phosphorylated protein kinase Cα, as well as reduced abundance of lysosomal membrane proteins such as LAMP1. Testing activators of lysosomal function showed that specific phorbol esters, such as PEP005, reduced α-synuclein and phosphorylated protein kinase Cα levels while increasing LAMP1 abundance. Interestingly, the reduction in α-synuclein occurred through proteasomal degradation. PEP005 delivery to mouse striatum also decreased α-synuclein production in vivo. Induced pluripotent stem cell-derived dopaminergic cultures reveal a signature in patients with YOPD who have no known Parkinson's disease-related mutations, suggesting that there might be other genetic contributions to this disorder. This signature was normalized by specific phorbol esters, making them promising therapeutic candidates.

Detection and Quantification of GPCR mRNA: An Assessment and Implications of Data from High-Content Methods.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and targets for approved drugs. The analysis of GPCR expression is, thus, important for drug discovery and typically involves messenger RNA (mRNA)-based methods. We compared transcriptomic complementary DNA (cDNA) (Affymetrix) microarrays, RNA sequencing (RNA-seq), and quantitative polymerase chain reaction (qPCR)-based TaqMan arrays for their ability to detect and quantify expression of endoGPCRs (nonchemosensory GPCRs with endogenous agonists). In human pancreatic cancer-associated fibroblasts, RNA-seq and TaqMan arrays yielded closely correlated values for GPCR number (∼100) and expression levels, as validated by independent qPCR. By contrast, the microarrays failed to identify ∼30 such GPCRs and generated data poorly correlated with results from those methods. RNA-seq and TaqMan arrays also yielded comparable results for GPCRs in human cardiac fibroblasts, pancreatic stellate cells, cancer cell lines, and pulmonary arterial smooth muscle cells. The magnitude of mRNA expression for several Gq/11-coupled GPCRs predicted cytosolic calcium increase and cell migration by cognate agonists. RNA-seq also revealed splice variants for endoGPCRs. Thus, RNA-seq and qPCR-based arrays are much better suited than transcriptomic cDNA microarrays for assessing GPCR expression and can yield results predictive of functional responses, findings that have implications for GPCR biology and drug discovery.

A Simple and Reliable Cooling Approach for the Cryopreservation of Hematopoietic Stem Cells: the Passive Cooling Rate-controlled Technique.

BACKGROUND: Cryopreservation of hematopoietic stem cells (HSCs) has widely been used in stem cell transplantation and cellular therapy treating various human diseases. However, the current conventional cooling approach for the cryopreservation of HSCs has the following potential problems: (1) requirement of a very expensive computer-programmed liquid nitrogen freezer (LNF) for the cooling rate control, (2) a large consumption of liquid nitrogen, (3) periodic breakdown of the LNF due to the mechanical failure of the liquid nitrogen valves (i.e., magnetic-solenoid valves) inside the LNF, and (4) constant monitoring of the LNF operation during the HSCs cooling process. OBJECTIVE: To test and evaluate a simple and reliable approach for the cryopreservation of HSCs using the passive cooling technique. MATERIALS AND METHODS: A passive cooling-rate-controlled device (PCD) was developed and used to cryopreserve HSCs. The PCD is inexpensive, simple, and user-friendly, which needs only the minimum maintenance and no consumption of liquid nitrogen. The PCD was compared to the LNF for the cryopreservation of HSCs in the present study through experiments. The cell viability and functionality were evaluated after cryopreservation. RESULTS: In comparison with the LNF method, the PCD approach enabled high cell viability/survival, recovery rate, and functionality after cryopreservation processes. CONCLUSION: The PCD offers a cost-effective, simple, and reliable approach for the optimal cryopreservation of HSCs.

GPCRomics: An Approach to Discover GPCR Drug Targets.

G protein-coupled receptors (GPCRs) are targets for ∼35% of approved drugs but only ∼15% of the ∼800 human GPCRs are currently such targets. GPCRomics, the use of unbiased, hypothesis-generating methods [e.g., RNA-sequencing (RNA-seq)], with tissues and cell types to identify and quantify GPCR expression, has led to the discovery of previously unrecognized GPCRs that contribute to functional responses and pathophysiology and that may be therapeutic targets. The combination of GPCR expression data with validation studies (e.g., signaling and functional activities) provides opportunities for the discovery of disease-relevant GPCR targets and therapeutics. Here, we review insights from GPCRomic approaches, gaps in knowledge, and future directions by which GPCRomics can advance GPCR biology and the discovery of new GPCR-targeted drugs.

GPR68: An Emerging Drug Target in Cancer.

GPR68 (or ovarian cancer G protein-coupled receptor 1, OGR1) is a proton-sensing G-protein-coupled receptor (GPCR) that responds to extracellular acidity and regulates a variety of cellular functions. Acidosis is considered a defining hallmark of the tumor microenvironment (TME). GPR68 expression is highly upregulated in numerous types of cancer. Emerging evidence has revealed that GPR68 may play crucial roles in tumor biology, including tumorigenesis, tumor growth, and metastasis. This review summarizes current knowledge regarding GPR68-its expression, regulation, signaling pathways, physiological roles, and functions it regulates in human cancers (including prostate, colon and pancreatic cancer, melanoma, medulloblastoma, and myelodysplastic syndrome). The findings provide evidence for GPR68 as a potentially novel therapeutic target but in addition, we note challenges in developing drugs that target GPR68.

GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets.

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.

GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

The microenvironment of pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense fibrotic stroma (desmoplasia) generated by pancreatic cancer-associated fibroblasts (CAFs) derived from pancreatic stellate cells (PSCs) and pancreatic fibroblasts (PFs). Using an unbiased GPCRomic array approach, we identified 82 G-protein-coupled receptors (GPCRs) commonly expressed by CAFs derived from 5 primary PDAC tumors. Compared with PSCs and PFs, CAFs have increased expression of GPR68 (a proton-sensing GPCR), with the results confirmed by immunoblotting, The Cancer Genome Atlas data, and immunohistochemistry of PDAC tumors. Co-culture of PSCs with PDAC cells, or incubation with TNF-α, induced GPR68 expression. GPR68 activation (by decreasing the extracellular pH) enhanced IL-6 expression via a cAMP/PKA/cAMP response element binding protein signaling pathway. Knockdown of GPR68 by short interfering RNA diminished low pH-induced production of IL-6 and enhancement of PDAC cell proliferation by CAF conditioned media. CAFs from other gastrointestinal cancers also express GPR68. PDAC cells thus induce expression by CAFs of GPR68, which senses the acidic microenvironment, thereby increasing production of fibrotic markers and IL-6 and promoting PDAC cell proliferation. CAF-expressed GPR68 is a mediator of low-pH-promoted regulation of the tumor microenvironments, in particular to PDAC cell-CAF interaction and may be a novel therapeutic target for pancreatic and perhaps other types of cancers.-Wiley, S. Z., Sriram, K., Liang, W., Chang, S. E., French, R., McCann, T., Sicklick, J., Nishihara, H., Lowy, A. M., Insel, P. A. GPR68, a proton-sensing GPCR, mediates interaction of cancer-associated fibroblasts and cancer cells.

Three-dimensional digitalized virtual planning for saphenous artery flap: a pilot study.

OBJECTIVE: Since the 1970s, research and applications on flap and muscle flap had solved many problems in microsurgical reconstruction. However, the traditional flap design is completely dependent on two-dimensional (2D) images. The purpose of this study was to discuss the methods in the visualization of saphenous artery flap by digitalized technique and its applications by digitalized technique. METHODS: Two adult fresh cadaver specimens, one male and one female, were subject to radiographic computerized tomography (CT) scanning before and after perfused with lead oxide-gelatine mixture, whose collimation are 0.625 mm (120 kV, 110 mA, 512 × 512 matrix). Through Amira 5.4.1 software, the 2D images in DICOM format were transformed into the 3D models of the entire region. The structures of saphenous artery were observed and the digitized visible models of saphenous artery flap were established through three-dimensional (3D) computerized reconstructions methods from these data using Amira 5.4.1 software. Next six cases of soft-tissue defects of the tibia region, involving the exposure bones underwent contrast-enhanced CT angiography of lower limbs utilizing a 64-row multi-slice spiral CT after median cubital vein injection with Ultravist (3.5 ml/s). 2D images from these data in DICOM format were transformed into computer. The structures of saphenous artery flap were observed and measured using Amira 5.4.1 software. Then, all cases were treated by saphenous artery flap. RESULTS: The 3D reconstructed visible models established from these datasets perfectly displayed the saphenous artery flap anatomy. In six cases, the main trunk and branched of the blood vessels in the designed flap were consistent with the surgical findings. The starting point of the saphenous artery to the average distance of the knee clearance were 119.2 ± 9.6 mm, the average diameter of the saphenous artery from the starting point were 1.5 ± 0.3 mm. The range of flap was 8.0 × 5.0 cm to 20.0 × 8.0 cm. All flaps survived well. After 8-24 months' follow-up the knee flexion was 120-140°, the straight 0-10°. There was no case appeared incision infection. CONCLUSIONS: The preoperative use of 3D digitalized virtual planning for the saphenous artery flap improves the surgical accuracy, decreases the operation time and increases the survival rate of the flap.

[Expression and function of T-type α1H Ca(2)(+) channels in the rat model of Hirschsprung disease].

OBJECTIVE: To investigate the role of T-type α1H Ca(2+) channels(Cav 3.2) in the pathogenesis of Hirschsprung disease(HD). METHOD: Eighty neonatal SD rats 6 to 8 days of age were randomly divided into 2 groups, 40 in each. Microinjector catheters were carefully placed into the bowl of one group, and 0.2% benzalkonium chloride (BAC) solution was injected to establish HD rat model. Control group was treated with saline instead of benzalkonium chloride. At postoperative 4, 6 and 8 weeks, ten rats were sacrificed randomly and examined through general observation, histopathological observation and immunofluorescent staining of PGP 9.5 to identify the animal models. Meanwhile, the distribution of Cav 3.2 in abnormal colon of HD rat model was studied by immunohistochemical staining, Western blot, and the co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Besides, function of Cav 3.2 was surveyed with the model rats tensile force and frequency of colonic muscle in vitro. RESULT: After 8 weeks of BAC treatment, the rat model was successfully established according to the results of histopathological and immunofluorescent staining, which showed decrease or lack of ganglion cells within the area of BAC treatment. The distribution of Cav 3.2 was detected by immunohistochemical staining. In the normal colon, Cav 3.2 were mainly distributed between circular muscle and longitudinal muscle, and showed continuous distribution. However, in the narrow segment of HD rat model, the distribution of Cav 3.2 was decreased significantly, and its continuity was destroyed . The results of Western blot were quite consistent with immunohistochemistry staining, in the narrow segment of HD rat model, the expression of Cav 3.2 was decreased gradually after the BAC treatment, especially at postoperative 8 weeks. The relative expression of Cav 3.2 of the control group was 0.63±0.06, 0.62±0.09, 0.63±0.06 at postoperative 4, 6 and 8 weeks respectively. While that of HD group was 0.63±0.06, 0.38±0.06, 0.26±0.07 respectively, which was significantly lower as compared with that of control group at postoperative 6 and 8 weeks respectively (t-value 5.27 and 8.63 respectively, both P<0.05). The co-localization of Cav 3.2 and c-kit was studied by double immunofluorescent staining. Similarly, compared with control groups, the co-localization of Cav 3.2 and c-kit were reduced obviously or vanished in the narrow segment of HD rat model. In motility studies, the control rats show cyclic depolarization regularly. However, the cyclic depolarization largely disappeared in model rats. Besides, on the premise of cyclic depolarization in control muscle strips, when we added ZnCl2, known to inhibit Cav 3.2 selectively among three T-channel isoforms, into tissue chambers, the pattern of muscle contractions appear similar to HD model rats. CONCLUSION: The abnormal alteration of Cav 3.2 probably mediate the functional change of interstitial cells of cajal in the HD, and finally induce the intestinal dysfunction of HD.