MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

BACKGROUND: Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. METHODS: In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. RESULTS: The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. CONCLUSIONS: This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

Systems-Based Physical Assessments: Earlier Detection of Clinical Deterioration and Reduced Mortality.

BACKGROUND: Despite efforts to improve early detection of deterioration in a patient's condition, delays in activating the rapid response team remain common. OBJECTIVES: To evaluate delays in activating the rapid response team and the occurrence of serious adverse events before and after implementation of a quality improvement initiative aimed at nurses' performing systems-based physical assessments. METHODS: A retrospective observational cohort design was used to evaluate all patients who had a rapid response team activation during the study period. RESULTS: A total of 1080 patients were included in the analysis: 536 patients before the quality improvement initiative and 544 patients after the quality improvement initiative. The delay in activating the rapid response team decreased from 11.7 hours in the before group to 9.6 hours in the after group (P < .001). In the after group, fewer patients were transferred to the intensive care unit (36% vs 41%, P = .02) and those who were transferred had 3.58 times greater odds of death than those who stayed at the same level of care. The after group had a 44% reduction in the odds of mortality compared with the before group. CONCLUSIONS: When nurses focus on conducting a systems-based physical assessment early in their shift, delays in recognizing a patient's deteriorating condition are reduced, fewer patients are admitted to the intensive care unit, and mortality is significantly reduced.

HSD3B1 Expression Is Upregulated by Interleukin 4 in HT-29 Colon Cancer Cells via Multiple Signaling Pathways.

3ß-Hydroxysteroid dehydrogenase/isomerase is essential for the synthesis of active steroid hormones. Interleukin 4 (IL4) induces the expression of HSD3B1 in various human cancer cell lines. Here, we demonstrated that administration of IL4 to an HT-29 colon cancer cell line induced high expression of HSD3B1 at the mRNA and protein levels. In the HT-29 cells, IL4 stimulated the activity of signal transducer and activator of transcription 6 (STAT6) and promoted its binding to the STAT6-binding site in the HSD3B1 promoter. The STAT6 inhibitor significantly suppressed HSD3B1 induction by IL4 in a dose-dependent manner. Moreover, inhibition of the PI3-kinase/AKT pathway strongly suppressed the IL4-induced HSD3B1 expression. Glycogen synthase kinase 3 (GSK3), a downstream target of AKT, had a stimulatory effect on the IL4-induced HSD3B1 expression. However, IL4 stimulated the phosphorylation of AKT, which inhibited the GSK3 activity at the early stage. Hence, GSK3 potentiated the HSD3B1 levels at the late stage of the IL4 stimulation. Additionally, inhibitors of mitogen-activated protein kinases (MAPKs), ERK1/2 and p38, but not of JNK, partly reduced the HSD3B1 expression following the IL4 stimulation. We further demonstrated that IL4 potently promoted steroid synthesis. Our results indicate that IL4 induces HSD3B1 expression via multiple signaling pathways in HT-29 cells and may play a role in the regulation of steroid synthesis.

A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan.

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Physical assessment competencies for nurses: A quality improvement initiative.

As the only healthcare providers caring for hospitalized patients every hour of every day, nurses have a responsibility to keep patients safe. Physical assessment is a basic but essential nursing skill that fosters patient safety. Assessing a patient's current status enables nurses to recognize early patient deterioration. Contemporary nursing practice relies on vital signs and technology to aid in the detection of patient deterioration. The aim is to describe the Methodist Proficient Assessment Competency (MPAC© ) quality improvement initiative. Surveys and directly observed patient assessment data were used to evaluate attitudes and practices. One hundred and seventy-nine pre-MPAC audits were conducted, followed by 1391 post-MPAC audits. Pre- compared with post-MPAC audits showed significant improvements in complete physical assessments (78% vs. 94%; p < .001), timeliness (within 4 h; 64% vs. 91%; p < .001) and accuracy (67% vs. 95%; p < .001) of documentation. In conclusion, nurses have a responsibility to quickly identify changes in a patient's condition and intervene to prevent serious adverse events. Taking the needed time to perform a full physical assessment at the beginning of the shift along with timely and accurate documentation, allows nurses to acquire the knowledge they need to establish a patient's current clinical status and usual behaviors, thereby facilitating early recognition of subtle changes that could indicate deterioration.

Specific Plasticity Loci and Their Synergism Mediate Operant Conditioning.

Despite numerous studies examining the mechanisms of operant conditioning (OC), the diversity of OC plasticity loci and their synergism have not been examined sufficiently. In the well-characterized feeding neural circuit of Aplysia, in vivo and in vitro appetitive OC increases neuronal excitability and electrical coupling among several neurons leading to an increase in expression of ingestive behavior. Here, we used the in vitro analog of OC to investigate whether OC reduces the excitability of a neuron, B4, whose inhibitory connections decrease expression of ingestive behavior. We found OC decreased the excitability of B4. This change appeared intrinsic to B4 because it could be replicated with an analog of OC in isolated cultures of B4 neurons. In addition to changes in B4 excitability, OC decreased the strength of B4's inhibitory connection to a key decision-making neuron, B51. The OC-induced changes were specific without affecting the excitability of another neuron critical for feeding behavior, B8, or the B4-to-B8 inhibitory connection. A conductance-based circuit model indicated that reducing the B4-to-B51 synapse, or increasing B51 excitability, mediated the OC phenotype more effectively than did decreasing B4 excitability. We combined these modifications to examine whether they could act synergistically. Combinations including B51 synergistically enhanced feeding. Taken together, these results suggest modifications of diverse loci work synergistically to mediate OC and that some neurons are well suited to work synergistically with plasticity in other loci.SIGNIFICANCE STATEMENT The ways in which synergism of diverse plasticity loci mediate the change in motor patterns in operant conditioning (OC) are poorly understood. Here, we found that OC was in part mediated by decreasing the intrinsic excitability of a critical neuron of Aplysia feeding behavior, and specifically reducing the strength of one of its inhibitory connections that targets a key decision-making neuron. A conductance-based computational model indicated that the known plasticity loci showed a surprising level of synergism to mediate the behavioral changes associated with OC. These results highlight the importance of understanding the diversity, specificity and synergy among different types of plasticity that encode memory. Also, because OC in Aplysia is mediated by dopamine (DA), the present study provides insights into specific and synergistic mechanisms of DA-mediated reinforcement of behaviors.

Microvascular Fluid Resuscitation in Circulatory Shock.

The microcirculation is responsible for blood flow regulation and red blood cell distribution throughout individual organs. Patients with circulatory shock have acute failure of the cardiovascular system in which there is insufficient delivery of oxygen to meet metabolic tissue requirements. All subtypes of shock pathophysiology have a hypovolemic component. Fluid resuscitation guided by systemic hemodynamic end points is a common intervention. Evidence shows that microcirculatory shock persists even after optimization of macrocirculatory hemodynamics. The ability for nurses to assess the microcirculation at the bedside in real-time during fluid resuscitation could lead to improved algorithms designed to resuscitate the microcirculation.

An extended EPQ-based problem with a discontinuous delivery policy, scrap rate, and random breakdown.

In real supply chain environments, the discontinuous multidelivery policy is often used when finished products need to be transported to retailers or customers outside the production units. To address this real-life production-shipment situation, this study extends recent work using an economic production quantity- (EPQ-) based inventory model with a continuous inventory issuing policy, defective items, and machine breakdown by incorporating a multiple delivery policy into the model to replace the continuous policy and investigates the effect on the optimal run time decision for this specific EPQ model. Next, we further expand the scope of the problem to combine the retailer's stock holding cost into our study. This enhanced EPQ-based model can be used to reflect the situation found in contemporary manufacturing firms in which finished products are delivered to the producer's own retail stores and stocked there for sale. A second model is developed and studied. With the help of mathematical modeling and optimization techniques, the optimal run times that minimize the expected total system costs comprising costs incurred in production units, transportation, and retail stores are derived, for both models. Numerical examples are provided to demonstrate the applicability of our research results.

A chemical compound commonly used to inhibit PKR, {8-(imidazol-4-ylmethylene)-6H-azolidino[5,4-g] benzothiazol-7-one}, protects neurons by inhibiting cyclin-dependent kinase.

Activation of the double-stranded RNA-dependent protein kinase (PKR) has been implicated in the pathogenesis of several neurodegenerative diseases. We find that a compound widely used as a pharmacological inhibitor of this enzyme, referred to as PKR inhibitor (PKRi), {8-(imidazol-4-ylmethylene)-6H-azolidino[5,4-g]benzothiazol-7-one}, protects against the death of cultured cerebellar granule and cortical neurons. PKRi also prevents striatal neurodegeneration and improves behavioral outcomes in a chemically induced mouse model of Huntington's disease. Surprisingly, PKRi fails to block the phosphorylation of eIF2alpha, a downstream target of PKR, and does not reduce the autophosphorylation of PKR enzyme immunoprecipitated from neurons. Furthermore, neurons lacking PKR are fully protected from apoptosis by PKRi, demonstrating that neuroprotection by this compound is not mediated by PKR inhibition. Using in vitro kinase assays we investigated whether PKRi affects any other protein kinase. These analyses demonstrated that PKRi has no major inhibitory effect on pro-apoptotic kinases such as the c-Jun N-terminal kinases, the p38 MAP kinases and the death-associated protein kinases, or on other kinases including c-Raf, MEK1, MKK6 and MKK7. PKRi does, however, inhibit the activity of certain cyclin-dependent kinases (CDKs), including CDK1, CDK2 and CDK5 both in vitro and in low potassium-treated neurons. Consistent with its inhibitory action on mitotic CDKs, the treatment of HT-22 and HEK293T cell lines with PKRi sharply reduces the rate of cell cycle progression. Taken together with the established role of CDK activation in the promotion of neurodegeneration, our results suggest that PKRi exerts its neuroprotective action by inhibiting CDK.

False-positive analysis of functional MRI during simulated deep brain stimulation: a phantom study.

PURPOSE: To investigate the false-positive activations/deactivations in functional MRI (fMRI) of deep brain stimulation (DBS) using a phantom. MATERIALS AND METHODS: fMRI experiments were performed on a 1.5T scanner using a single-shot gradient-echo echo-planar imaging (GE-EPI) sequence (TR/TE/FA = 6000 msec/60 msec/90 degrees ) on an agar-gel phantom inserted with DBS electrodes. During the experimental blocks, two-second stimuli were delivered during the interscan waiting time (ISWT), which was adjusted by changing the number of slices acquired within the TR (3500 msec with 30 slices and 5160 msec with 10 slices). Data were analyzed using SPM2 software, and the false-positive voxels were detected with five different P-value thresholds. RESULTS: The number of false-positive voxels in experimental conditions had no significant differences from those in control conditions with either long or short ISWT, which increased with the P-value threshold from zero at P < 0.0001 to approximately 40 at P < 0.05. The pattern of increasing number of false-positive reactions along with P-value was similar between all conditions. CONCLUSION: False-positive findings from fMRI with similar experimental design can be well controlled with a statistical threshold of P < 0.001 or tighter. The short ISWT of 3500 msec did not increase false-positive reactions compared to the long ISWT of 5160 msec.

Histone deacetylase-related protein inhibits AES-mediated neuronal cell death by direct interaction.

Histone deacetylase-related protein (HDRP), an alternatively spliced and truncated form of histone deacetylase-9 that lacks a C-terminal catalytic domain, protects neurons from death. In an effort to understand the mechanism by which HDRP mediates its neuroprotective effect, we screened for proteins in the brain that interact with HDRP by using a yeast two-hybrid assay. One of the HDRP-interacting proteins identified in this screen was amino enhancer of split (AES), a 197-amino acid protein belonging to the Groucho family. Interaction between HDRP and AES was verified by in vitro binding assays, coimmunoprecipitation, and colocalization studies. To investigate the significance of the HDRP-AES association to the regulation of neuronal survival, we used cultured cerebellar granule neurons, which undergo apoptosis when treated with low potassium (LK) medium. We found that in contrast to HDRP, whose expression is markedly reduced by LK treatment, AES expression was not appreciably altered. Forced expression of AES in healthy neurons results in cell death, an action that is blocked by the coexpression of HDRP. AES is a truncated version of larger Groucho-related proteins, one of which is transducin-like enhancer of split (TLE)-1. We found that the expression of TLE1 is reduced in LK-treated neurons and the forced expression of TLE1 blocks LK-induced neuronal death as well as death induced by AES. Our results show that AES has apoptotic activity in neurons and suggest that neuroprotection by HDRP is mediated by the inhibition of this activity through direct interaction.

Inhibition of ATF-3 expression by B-Raf mediates the neuroprotective action of GW5074.

GW5074 a brain-permeable 3' substituted indolone, protects neurons from death in culture and in an in vivo paradigm of neurodegeneration. Using low potassium (LK) induced apoptosis of cerebellar granule neurons, we report here that the protective action of GW5074 is mediated through the activation of B-Raf. Over-expression of a kinase-dead form of B-Raf blocks the ability of GW5074 to neuroprotect, whereas over-expression of active forms of B-Raf protect even in the absence of GW5074. Although mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) are activated by GW5074, pharmacological inhibition of MEK-ERK signaling by U0126 or PD98059 does not reduce neuroprotection suggesting that B-Raf signals through a non-canonical signaling pathway. GeneChip microarray analyses identified activating transcription factor-3 (ATF-3) as a gene whose expression is induced by LK but that is negatively regulated by GW5074. Forced inhibition of ATF-3 expression using siRNA protects neurons against LK-induced apoptosis, whereas the over-expression of ATF-3 blocks GW5074-mediated neuroprotection. Not unexpectedly, expression of active B-Raf inhibits the apoptosis-associated increase in ATF-3 expression. We extended our work to include three other 3' substituted indolones - a commercially available inhibitor of RNA-dependent protein kinase and two novel compounds designated as SK4 and SK6. Like GW5074, RNA-dependent protein kinase inhibitor, SK4, and SK6 all inhibited c-Raf in vitro but activated B-Raf in neuronal cultures. All four compounds also inhibited ATF-3 expression. Taken together our results indicate that all four indolones mediate neuroprotection by a common mechanism which involves B-Raf activation, and that a downstream target of B-Raf is ATF-3.

Inhibition of neuronal apoptosis by the cyclin-dependent kinase inhibitor GW8510: identification of 3' substituted indolones as a scaffold for the development of neuroprotective drugs.

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3' substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3beta and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3' substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3' substituted indolone) as a neuroprotective compound, our findings identify the 3' substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.