Molecular Determinants of the Differential Modulation of Cav1.2 and Cav1.3 by Nifedipine and FPL 64176.

Nifedipine and FPL 64176 (FPL), which block and potentiate L-type voltage-gated Ca2+ channels, respectively, modulate Cav1.2 more potently than Cav1.3. To identify potential strategies for developing subtype-selective inhibitors, we investigated the role of divergent amino acid residues in transmembrane domains IIIS5 and the extracellular IIIS5-3P loop region in modulation of these channels by nifedipine and FPL. Insertion of the extracellular IIIS5-3P loop from Cav1.2 into Cav1.3 (Cav1.3+) reduced the IC50 of nifedipine from 289 to 101 nM, and substitution of S1100 with an A residue, as in Cav1.2, accounted for this difference. Substituting M1030 in IIIS5 to V in Cav1.3+ (Cav1.3+V) further reduced the IC50 of nifedipine to 42 nM. FPL increased current amplitude with an EC50 of 854 nM in Cav1.3, 103 nM in Cav1.2, and 99 nM in Cav1.3+V. In contrast to nifedipine block, substitution of M1030 to V in Cav1.3 had no effect on potency of FPL potentiation of current amplitude, but slowed deactivation in the presence and absence of 10 µM FPL. FPL had no effect on deactivation of Cav1.3/dihydropyridine-insensitive (DHPi), a channel with very low sensitivity to nifedipine block (IC50 ∼93 µM), but did shift the voltage-dependence of activation by ∼-10 mV. We conclude that the M/V variation in IIIS5 and the S/A variation in the IIIS5-3P loop of Cav1.2 and Cav1.3 largely determine the difference in nifedipine potency between these two channels, but the difference in FPL potency is determined by divergent amino acids in the IIIS5-3P loop.

RyR2 regulates store-operated Ca2+ entry, phospholipase C activity, and electrical excitability in the insulinoma cell line INS-1.

The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 µM carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates ß-cell electrical activity by controlling Cav current density and SK channel activation.

Effectiveness of a COVID-19 Preventive Sequestration Strategy: Deployment of the Aircraft Carrier USS Ronald Reagan (CVN-76).

OBJECTIVES: Develop and evaluate a pre-deployment sequestration (PDS) protocol to prevent SARS-CoV-2 cases on board the USS RONALD REAGAN (CVN-76). METHODS: The USS RONALD REAGAN includes a crew of approximately 3,000 Sailors and an embarked Air Wing of 2,000 personnel. The PDS was conducted in three waves of 14-day strict quarantines during the months of April and May 2020. Sailors were cleared to board the ship with two negative rtPCR tests at days 14 and 16. The ship was sanitized prior to Wave 1 boarding. RESULTS: From March 1, 2020 through May 31, 2020, a total of 51 SARS-CoV-2 positive cases were detected. During the three waves of PDS, 28 Sailors were found to be positive on exit testing (14, 11, and 3, respectively); no cases were found among the Air Wing. During the first 90 days at sea, no SARS-CoV-2 cases were detected among any of the embarked personnel. CONCLUSIONS: Although resource-intensive, the PDS protocol implemented for USS RONALD REAGAN resulted in a COVID-free ship during a global pandemic with unprecedented scope. Elements of this pandemic PDS protocol may be useful in other highly risk-averse environments with no tolerance for COVID-19 infections.

RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1.

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic ß-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced IP3 receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO and IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

Regulation of cAMP accumulation and activity by distinct phosphodiesterase subtypes in INS-1 cells and human pancreatic ß-cells.

Pancreatic ß-cells express multiple phosphodiesterase (PDE) subtypes, but the specific roles for each in ß-cell function, particularly in humans, is not clear. We evaluated the cellular role of PDE1, PDE3, and PDE4 activity in the rat insulinoma cell line INS-1 and in primary human ß-cells using subtype-selective PDE inhibitors. Using a genetically encoded, FRET-based cAMP sensor, we found that the PDE1 inhibitor 8MM-IBMX, elevated cAMP levels in the absence of glucose to a greater extent than either the PDE3 inhibitor cilostamide or the PDE4 inhibitor rolipram. In 18 mM glucose, PDE1 inhibition elevated cAMP levels to a greater extent than PDE3 inhibition in INS-1 cells, while PDE4 inhibition was without effect. Inhibition of PDE1 or PDE4, but not PDE3, potentiated glucose-stimulated insulin secretion in INS-1 cells. PDE1 inhibition, but not PDE3 or PDE4 inhibition, reduced palmitate-induced caspase-3/7 activation, and enhanced CREB phosphorylation in INS-1 cells. In human ß-cells, only PDE3 or PDE4 inhibition increased cAMP levels in 1.7 mM glucose, but PDE1, PDE3, or PDE4 inhibition potentiated cAMP levels in 16.7 mM glucose. Inhibition of PDE1 or PDE4 increased cAMP levels to a greater extent in 16.7 mM glucose than in 1.7 mM glucose in human ß-cells. In contrast, elevation of cAMP levels by PDE3 inhibition was not different at these glucose concentrations. PDE1 inhibition also potentiated insulin secretion from human islets, suggesting that the role of PDE1 may be conserved between INS-1 cells and human pancreatic ß-cells. Our results suggest that inhibition of PDE1 may be a useful strategy to potentiate glucose-stimulated insulin secretion, and to protect ß-cells from the toxic effects of excess fatty acids.

Recurrent pure mucinous carcinoma of the breast with mediastinal great vessel invasion: HER-2/neu confers aggressiveness.

Mucinous carcinoma of the breast, also known as colloid carcinoma, is a less common variant of breast cancer constituting less than five per cent of breast cancers. We report the case of a 42-year-old premenopausal female who presented with a palpable chest wall recurrence 4 years after simple mastectomy, axillary node dissection, and TRAM flap reconstruction for pure mucinous carcinoma. The recurrent neoplasm was a pure mucinous carcinoma and was found to be invading the mediastinum into the great vessels. The tumor was estrogen receptor positive, progesterone receptor negative, and HER-2/neu positive, which is an unusual finding for mucinous carcinoma. The fact that this tumor demonstrated HER-2/neu positivity may explain the uncharacteristic aggressive nature of this normally indolent type of breast tumor. To our knowledge, this is the first reported case of any mucinous breast cancer invading the mediastinal great vessels and its subsequent en-bloc resection.

Seat belt trauma: pectoralis muscle rupture and delayed mesh repair.

To our knowledge, this is the first reported case of a seat belt-related rupture of the pectoralis major muscle and its successful delayed repair using mesh. We report a case of a 34-year-old white man who sustained a right pectoralis major muscle rupture from a seat belt during a motor vehicle crash. The patient presented to us 2 years after the injury. We introduce a technique using mesh that results in a successful repair of a cosmetically disfiguring chest wall defect.

Inferior vena cava filter migration to right ventricle with destruction of tricuspid valve: a case report.

Inferior vena cava filter migration is an uncommon event. Temporary inferior vena cava filters offer protection against pulmonary embolism in the trauma patient in whom anticoagulation is contraindicated. We present the case of a 53-year-old man who suffered a lower extremity injury, which left him unable to walk for an extended period of time. The patient developed a deep venous thrombosis in the early postoperative course and decision was made to place a retrievable inferior vena cava (IVC) filter. One week later the IVC filter had migrated to the right ventricle and destroyed the tricuspid valve. Although there are a limited number of cases describing the migration of IVC filters to the heart, there have been no cases in the literature, to our knowledge, where an IVC filter has destroyed the tricuspid valve and required valve replacement.

Selective Inhibition of STAT3 Phosphorylation Using a Nuclear-Targeted Kinase Inhibitor.

The discovery of compounds that selectively modulate signaling and effector proteins downstream of EGFR could have important implications for understanding specific roles for pathway activation. A complicating factor for receptor tyrosine kinases is their capacity to be translocated to the nucleus upon ligand engagement. Once localized in subcellular compartments like the nucleus, the roles for EGFR take on additional features, many of which are still being revealed. Additionally, nuclear localization of EGFR has been implicated in downstream events that have significance for therapy resistance and disease progression. The challenges to addressing the differential roles for EGFR in the nucleus motivated experimental approaches that can selectively modulate its subcellular function. By adding modifications to the established EGFR kinase inhibitor gefitinib, an approach to small molecule conjugates with a unique nuclear-targeting peptoid sequence was tested in both human and murine breast tumor cell models for their capacity to inhibit EGF-stimulated activation of ERK1/2 and STAT3. While gefitinib alone inhibits both of these downstream effectors, data acquired here indicate that compartmentalization of the gefitinib conjugates allows for pathway specific inhibition of STAT3 while not affecting ERK1/2 signaling. The inhibitor conjugates offered a more direct route to evaluate the role of EGF-stimulated epithelial-to-mesenchymal transition in these breast cancer cell models. These conjugates revealed that STAT3 activation is not involved in EGF-induced EMT, and instead utilization of the cytoplasmic MAP kinase signaling pathway is critical to this process. This is the first example of a conjugate kinase inhibitor capable of partitioning to the nucleus and offers a new approach to enhancing kinase inhibitor specificity.

Laparoscopic resection of metastatic mucosal melanoma causing jejunal intussusception.

A 47-year-old female was seen in an outpatient surgical setting for intermittent obstructive symptoms and abdominal pain. Computed tomography scan and upper GI series showed typical findings of intussusception. Laparoscopic-assisted small bowel resection was carried out. Histologic diagnosis revealed malignant melanoma. The patient had a history of right maxillary sinus melanoma greater than 12 years before presentation. We report the first case of a jejunojejunal intussusception caused by metastatic sinonasal melanoma, and its subsequent laparoscopic-assisted resection.

Can intravenous lidocaine decrease postsurgical ileus and shorten hospital stay in elective bowel surgery? A pilot study and literature review.

BACKGROUND: This study examined whether systemic infusion of lidocaine, a local anesthetic with anti-inflammatory properties, can decrease surgical pain, length of postsurgical ileus, and hospital stay. METHODS: Twenty-two patients at a community hospital were randomized into 2 groups. Subjects were allocated to receive either lidocaine or a placebo infusion for the first 24 hours after surgery. RESULTS: Patients in the lidocaine group appeared to report less pain as reflected by a decrease in overall visual analogue scale pain scores 24 hours after surgery. The return of flatus after surgery was not considered significant (lidocaine 68.2 +/- 9.7 hours vs placebo 86.9 +/- 13.6 hours; P = .2802). The return of bowel movement after surgery was considered significant (lidocaine 88.3 +/- 6.08 hours vs placebo group 116 +/- 10.1 hours; P = .0286). The lidocaine group was discharged by mean day 3.76 +/- .24 versus placebo at mean day 4.93 +/- .42; P = .0277. CONCLUSIONS: Patients in the lidocaine group had bowel movements >24 hours earlier than those in the placebo group and were discharged earlier.