A Novel Flavi-like Virus in Alfalfa (Medicago sativa L.) Crops along the Snake River Valley

Alfalfa is an important perennial forage crop in Idaho supporting dairy and cattle industries that is typically grown in the same field for as many as 4 years. Alfalfa stands of different ages were subjected to screening for viruses using high-throughput sequencing and RT-PCR. The two most common viruses found were alfalfa mosaic virus and bean leafroll virus, along with Medicago sativa amalgavirus, two alphapartitiviruses, and one deltapartitivirus. Additionally, a new flavi-like virus with an unusual genome organization was discovered, dubbed Snake River alfalfa virus (SRAV). The 11,745 nt, positive-sense (+) RNA genome of SRAV encodes a single 3835 aa polyprotein with only two identifiable conserved domains, an RNA-dependent RNA polymerase (RdRP) and a predicted serine protease. Notably, unlike all +RNA virus genomes in the similar size range, the SRAV polyprotein contained no predicted helicase domain. In the RdRP phylogeny, SRAV was placed inside the flavi-like lineage as a sister clade to a branch consisting of hepaci-, and pegiviruses. To the best of our knowledge, SRAV is the first flavi-like virus identified in a plant host. Although commonly detected in alfalfa crops in southern Idaho, SRAV sequences were also amplified from thrips feeding in alfalfa stands in the area, suggesting a possible role of Frankliniella occidentalis in virus transmission.     

Vesicular nucleotide transporter is a molecular target of eicosapentaenoic acid for neuropathic and inflammatory pain treatment

Eicosapentaenoic acid (EPA), an omega-3 (ω-3) polyunsaturated fatty acid, is an essential nutrient that exhibits antiinflammatory, neuroprotective, and cardiovascular-protective activities. Although EPA is used as a nutrient-based pharmaceutical agent or dietary supplement, its molecular target(s) is debatable. Here, we showed that EPA and its metabolites strongly and reversibly inhibit vesicular nucleotide transporter (VNUT), a key molecule for vesicular storage and release of adenosine triphosphate (ATP) in purinergic chemical transmission. In vitro analysis showed that EPA inhibits human VNUT-mediated ATP uptake at a half-maximal inhibitory concentration (IC₅₀) of 67 nM, acting as an allosteric modulator through competition with Cl⁻. EPA impaired vesicular ATP release from neurons without affecting the vesicular release of other neurotransmitters. In vivo, VNUT^−/− mice showed a delay in the onset of neuropathic pain and resistance to both neuropathic and inflammatory pain. EPA potently attenuated neuropathic and inflammatory pain in wild-type mice but not in VNUT^−/− mice without affecting the basal nociception. The analgesic effect of EPA was canceled by the intrathecal injection of purinoceptor agonists and was stronger than that of existing drugs used for neuropathic pain treatment, with few side effects. Neuropathic pain impaired insulin sensitivity in previous studies, which was improved by EPA in the wild-type mice but not in the VNUT^−/− mice. Our results showed that VNUT is a molecular target of EPA that attenuates neuropathic and inflammatory pain and insulin resistance. EPA may represent a unique nutrient-based treatment and prevention strategy for neurological, immunological, and metabolic diseases by targeting purinergic chemical transmission.

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are essential nutrients that contain multiple double bonds. PUFAs can be classified into ω-3 and ω-6 depending on the position of the bonds. As humans cannot produce PUFAs, they must be acquired from the diet to maintain homeostasis. Omega-3 PUFAs, such as eicosapentaenoic acid (EPA), are abundantly present in fish and linseed oil and exhibit antiinflammatory, neuroprotective, and cardiovascular-protective activities via the competitive inhibition of cyclooxygenase (COX)-2 in eicosanoid production (1–3). Danish and Greenland Inuit epidemiological studies have reported that EPA reduces the risk of death after myocardial infarction (4, 5), and other studies have reported its influence on analgesia, neuroinflammatory disease (Parkinson’s disease, Alzheimer’s disease, and depression) improvement, platelet aggregation inhibition, decrease in blood triglyceride and glucose levels, and improved insulin resistance (1, 6–11). Omega-3 fatty acid supplementation in COVID-19 patients showed a beneficial effect in managing the cytokine storm (12). Conversely, omega-6 fatty acids, such as arachidonic acid, produce inflammatory eicosanoids and play central roles in the initial stage of inflammatory responses (13). Although arachidonic acid has also been reported to produce antiinflammatory metabolites, omega-6 PUFA-derived linoleate diols have a harmful effect and are biomarkers for severe COVID-19 infection (14). An omega-6 PUFA-enriched Western-style diet, which abundantly contains linoleate, causes neuropathy and chronic pain, but an omega-3 PUFA-enriched diet attenuates these pathological conditions (15).All therapeutic effects of EPA cannot be explained by COX-2 inhibition alone (16). Typically, COX-2 inhibitors (nonsteroidal antiinflammatory druga [NSAIDs]) are effective for inflammatory pain but ineffective for neuropathic pain (16). However, EPA significantly attenuates both inflammatory and neuropathic pain, which strongly suggests another important molecular target of EPA related to neuropathy (7, 8). Although chronic pain is coincidentally caused by inflammation and neuropathy, there is no therapeutic drug with few side effects to attenuate both inflammatory and neuropathic pain (17–20). In this situation, EPA may affect the key signaling molecule(s) in neurological, metabolic, and immunological functions.Purinergic chemical transmission is involved in neurological, metabolic, and immunological disruptions and functions, including neuropathic and inflammatory pain, depression, inflammation, increase in blood triglyceride and glucose levels, insulin resistance, and blood coagulation (21, 22). The released adenosine triphosphate (ATP) and degraded adenosine diphosphate (ADP) or adenosine binds to many types of purinoceptors that are intricately involved in biological and pathological processes. In pain perception, ATP and ADP bind to P2X and P2Y receptors and thereby exacerbate neuropathic and inflammatory pain (23). Adenosine binds to P1 receptors and thereby attenuates neuropathic and inflammatory pain (24). However, a vesicular nucleotide transporter (VNUT/SLC17A9) is localized in the secretory vesicles of neuronal, endocrine, and immune cells. It plays an essential role in vesicular ATP storage in a Δψ- and Cl^–-dependent manner in the purinergic chemical transmission, which leads to vesicular ATP release (25, 26). Thus, VNUT is a key molecule in the initiation of purinergic signaling for neurological, metabolic, and immunological disruptions and functions. Interestingly, the observed effects of the VNUT inhibitor and phenotypes of VNUT^−/− mice were consistent with the above-mentioned therapeutic effects of EPA (27–31). Therefore, we hypothesized that VNUT serves as a molecular target of EPA to attenuate neuropathic and inflammatory pain.Here, we demonstrated that a low concentration of EPA and its metabolites, but not docosahexaenoic acid (DHA), are potent and selective physiological inhibitors of vesicular ATP release via the blockade of purinergic chemical transmission, which improved neuropathic and inflammatory pain and insulin resistance. Furthermore, EPA is more effective for neuropathic and inflammatory pain and has fewer side effects than existing drugs.     

Transient receptor potential channels in rat renal microcirculation: actions of angiotensin II

BACKGROUND: This study assessed the calcium-activating mechanisms mediating glomerular arteriolar constriction by angiotensin II (Ang II). METHODS: Immunohistochemical and physiological studies were carried out, using antibody against transient receptor potential (TRP)-1 and an isolated perfused kidney model. RESULTS: Immunohistochemical experiments demonstrated that TRP-1 proteins were transcribed on both afferent and efferent arteriolar myocytes. In the first series of physiological experiments, Ang II (0.3 nmol/L) considerably constricted afferent (20.2 +/- 0.9 to 14.9 +/- 0.7 microm) and efferent arterioles (18.4 +/- 0.7 to 14.0 +/- 0.7 microm). The addition of nifedipine (1 micromol/L) restored decrements in afferent (to 20.0 +/- 0.8 microm) but not efferent arteriolar diameters. Further administration of SKF-96365 (100 micromol/L), a TRP channel blocker, reversed efferent arteriolar constriction (to 16.2 +/- 0.8 micromol/L). In the second group, although 2-aminoethoxydiphenyl borate (100 micromol/L), an inhibitor of inositol trisphosphate-induced calcium release (IP3CR), did not alter glomerular arteriolar diameters, it prevented Ang II-induced afferent arteriolar constriction and attenuated efferent arteriolar constriction (18.8 +/- 0.8 to 16.9 +/- microm). Subsequent removal of extracellular calcium abolished residual efferent arteriolar constriction (to 19.1 +/- 0.8 microm). CONCLUSIONS: Our data provide evidence that Ang II elicits IP3CR, possibly inducing a cellular response that activates voltage-dependent calcium channels on afferent arterioles. The present results suggest that Ang II-induced efferent arteriolar constriction involves IP3CR and calcium influx sensitive to SKF-96365.     

Percutaneous extracorporeal life support for treatment of fatal mechanical complications associated with acute myocardial infarction

Mechanical complications of acute myocardial infarction (AMI), such as free wall rupture, ventricular septal perforation (VSP), and mitral regurgitation due to papillary muscle rupture, are associated with high mortality rates. These complications result in extreme deterioration and increased risk of death in patients who do not receive timely resuscitation and surgical treatment. We studied the effectiveness of percutaneous extracorporeal life support (ECLS) for fatal mechanical AMI complications. Nine patients (7 men and 2 women, mean age 69 +/- 6 years) who suffered circulatory collapse refractory to conventional resuscitation were treated with ECLS. Circulatory collapse was caused by free wall rupture in 4 patients, VSP in 4, and mitral regurgitation due to papillary muscle rupture in 1. All patients were successfully resuscitated by ECLS and underwent surgical repair with conventional cardiopulmonary bypass. Eight patients required ECLS after surgery. Four of the 9 patients (2 with free wall rupture, 1 with VSP, and 1 with papillary muscle rupture) were successfully weaned from ECLS and were discharged. Three of the 4 survivors had no major complications, but the remaining survivor suffered neurological deficit. Four patients died while on devices. The duration of ECLS was from 13 to 167 h (mean 76 +/- 57 h) with a maximum bypass flow of 2.0 to 3.9 L/min (mean 2.9 +/- 0.6 L/min). There were no device-related complications during the support period. Total weaning rate was 56% (5/9), and survival was 44% (4/9). We conclude that ECLS can provide appropriate circulatory support during resuscitation and subsequent postoperative circulatory support for cardiovascular collapse associated with AMI complications.     

Laparoscopic correction of congenital portosystemic shunt in children

Congenital portosystemic shunt is a rare clinical entity that may progress to jaundice, severe encephalopathy, and pulmonary hypertension and require surgical correction or coil embolization. We present a novel approach to the management of children with congenital portosystemic shunt by means of a minimally invasive surgical technique. Congenital portosystemic shunts were identified between the superior mesenteric vein and inferior vena cava in case 1 and between the splenic vein and left renal vein in case 2. Both of them were successfully ligated by laparoscopic approach, and catheters were subsequently replaced to monitor portal venous pressure. The patients tolerated the procedure well, and short-term results were excellent. Laparoscopic ligation of congenital portosystemic shunt is technically feasible and less invasive to the management of patients with congenital portosystemic shunts, preventing late onset, life-threatening complications.     

Impact of the etiology of acute kidney injury on outcomes following liver transplantation: acute tubular necrosis versus hepatorenal syndrome

Acute kidney injury (AKI) at the time of liver transplantation (LT) has been associated with increased morbidity and mortality. In patients with potentially reversible renal dysfunction, predicting whether there will be sufficient return of native kidney function is sometimes difficult. Previous studies have focused mainly on the effect of the severity of renal dysfunction or the duration of pretransplant dialysis on posttransplant outcomes. We performed a retrospective analysis of patients who underwent LT at our center after Model for End-Stage Liver Disease-based allocation so that we could determine the impact of the etiology of AKI [acute tubular necrosis (ATN) versus hepatorenal syndrome (HRS)] on post-LT outcomes. The patients were stratified according to the severity of AKI at the time of LT as described by the Risk, Injury, Failure, Loss, and End-Stage Kidney Disease (RIFLE) classification: risk, injury, or failure. The RIFLE failure group was further subdivided according to the etiology of AKI: HRS or ATN. The patient survival and renal outcomes 1 and 5 years after LT were significantly worse for those with ATN. At 5 years, the incidence of chronic kidney disease (stage 4 or 5) was statistically higher in the ATN group versus the HRS group (56% versus 16%, P < 0.001). A multivariate analysis revealed that the presence of ATN at the time of LT was the only variable associated with higher mortality 1 year after LT (P < 0.001). Our study is the first to demonstrate that the etiology of AKI has the greatest impact on patient and renal outcomes after LT.     

Analgesic synergy between topical morphine and butamben in mice

Studies have revealed that lidocaine is an effective analgesic when applied topically to the tail of a mouse in the radiant heat tail-flick assay. In addition, the topical combination of lidocaine with morphine revealed synergistic interactions between the two drugs. In the current studies, we demonstrate that topical butamben, benzocaine, and bupivacaine are active in the radiant heat tail-flick assay. In this assay, topical lidocaine has a ceiling effect and displays a biphasic curve, with large doses markedly decreasing the responses almost to baseline levels. In contrast, butamben has an S-shape dose-dependent response in the assay and did not display a biphasic curve as seen with lidocaine, suggesting that topical butamben may have advantages over lidocaine. Both benzocaine and bupivacaine also showed dose-dependent analgesic activity in this model. Like lidocaine, butamben/morphine combinations displayed synergistic interactions. Indeed, the synergy appeared more prominent with a butamben/morphine combination. We also observed synergy between topical benzocaine and morphine. Although the bupivacaine/morphine combination was suggestive of synergy on isobolographic analysis, it did not achieve statistical significance. These studies indicate that a series of local anesthetics are all active topically in the radiant heat tail-flick assay in mice and that several interact synergistically with morphine. Of the local anesthetics tested, butamben seemed to have several pharmacological characteristics, alone and in combination with morphine, which suggest that it may be superior to the other local anesthetics. Together, these observations suggest that topical combinations of opioids and local anesthetics may prove clinically valuable. IMPLICATIONS: Topical administration of the opioid micro -agonist morphine and the sodium channel inhibitors butamben and benzocaine results in a synergistic interaction for antinociception in radiant heat tail-flick assay in mice, suggesting that the combination of these drugs will enhance rather than detract from the analgesia of either alone.     

Laparoscopic curative resection of pheochromocytomas

PURPOSE: Pheochromocytomas are relatively uncommon tumors whose operative resection has clear medical and technical challenges. While the safety and efficacy of laparoscopic adrenalectomy are relatively well documented, few studies with extended follow-up have been conducted to measure the success of the procedure for the most challenging of the adrenal tumors. In addition, several reports question the applicability of a minimally invasive approach for sizeable pheochromocytomas. The purpose of our investigation was to assess the outcomes of laparoscopic adrenalectomy for pheochromocytomas in the largest study to date when performed by experienced laparoscopic surgeons. METHODS: All pheochromocytomas removed by the authors from January 1995 to October 2004 were reviewed under an Institutional Review Board approved protocol. Eighty-five percent were documented in a prospective fashion. RESULTS: Eighty consecutive patients underwent laparoscopic resection of 81 pheochromocytomas. Seventy-nine were found in the adrenal (42 left, 35 right, 1 bilateral); 2 were extra-adrenal paragangliomas. Eight patients had multiple endocrine neoplasia syndrome. Two lesions were malignant. There were 48 females and 32 males with a mean age of 45 years (range, 15-79 years). Mean tumor size was 5.0 cm (range, 2-12.1 cm); 41 of these lesions were 5 cm in size or larger. Average operative time and blood loss were 169 minutes (range, 69-375 minutes) and 97 mL (range, 20-500 mL), respectively. Intraoperative hypertension (systolic blood pressure, >170 mm Hg) was reported in 53% of patients and hypotension (systolic blood pressure, <90 mm Hg) in 28% of patients. There were no conversions to open surgery. Mean length of stay was 2.3 days (range, 1-10 days). There were 6 perioperative morbidities (7.5%) and no mortalities. No patient required a blood transfusion. No recurrence of endocrinopathy has been documented at a mean follow-up of 21.4 months. CONCLUSION: Laparoscopic resection of pheochromocytomas, including large lesions, can be accomplished safely by experienced surgeons. A short hospital stay with minimal operative morbidity and eradication of endocrinopathy support the minimally invasive approach for adrenalectomy in the setting of pheochromocytoma.     

Role of ERK1/2 and p38 mitogen-activated protein kinases in the regulation of thrombospondin-1 by TGF-beta1 in rat proximal tubular cells and mouse fibroblasts

Thrombospondin-1 (TSP-1) inhibits angiogenesis and activates latent TGF-beta1, both of which are strongly associated with progression of renal disease. Recently, it was reported that Smad2 but not Smad3 regulates TSP-1 expression in response to TGF-beta1 in rat tubular epithelial cells as well as in mouse fibroblasts. This study investigated the role of ERK1/2 and p38 mitogen-activated protein kinases (MAPK). TGF-beta1 activated both ERK1/2 and p38 in the rat proximal tubular cell line NRK52E. Blocking ERK1/2 and p38 inhibited TGF-beta1-induced TSP-1 mRNA and protein expression. Next, the cross-talk between Smad2 and ERK1/2 or p38 was examined. Whereas blocking of ERK1/2 or p38 failed to inhibit TGF-beta1-induced Smad2 activation, inhibition of Smad2 by Smad7 overexpression inhibited the phosphorylation of ERK1/2 but not p38 in response to TGF-beta1. Similar results were observed using mouse fibroblasts from Smad2 knockout embryos, in that TGF-beta1 was able to activate p38 but not ERK1/2 in this cell line. In conclusion, TSP-1 expression is regulated by both ERK1/2 and p38 MAPK in rat proximal tubular cells and mouse fibroblasts in response to TGF-beta1. The ERK1/2 activation is dependent on Smad2 activation, whereas the p38 activation occurs independent of Smad2. Because TSP-1 is a major antiangiogenic molecule and an activator of TGF-beta1, this provides an important insight to the mechanism by which TGF-beta1 may mediate interstitial fibrosis and progressive renal disease.