Myofilament-based physiological regulatory compensation preserves diastolic function in failing hearts with severe Ca2+ handling deficits.

Severe dysfunction in cardiac muscle intracellular Ca2+ handling is a common pathway underlying heart failure. Here we used an inducible genetic model of severe Ca2+ cycling dysfunction by the targeted temporal gene ablation of the cardiac Ca2+ ATPase, SERCA2, in otherwise normal adult mice. In this model, in vivo heart performance was minimally affected initially, even though Serca2a protein was markedly reduced. The mechanism underlying the sustained in vivo heart performance in the weeks prior to complete heart pump failure and death is not clear and is important to understand. Studies were primarily focused on understanding how in vivo diastolic function could be relatively normal under conditions of marked Serca2a deficiency. Interestingly, data show increased cardiac troponin I (cTnI) serine 23/24 phosphorylation content in hearts upon Serca2a ablation in vivo. We report that hearts isolated from the Serca2-deficient mice retained near normal heart pump functional responses to ß-adrenergic stimulation. Unexpectedly, using genetic complementation models, in concert with inducible Serca2 ablation, data show that Serca2a-deficient hearts that also lacked the central ß-adrenergic signaling-dependent Serca2a negative regulator, phospholamban (PLN), had severe diastolic dysfunction that could still be corrected by ß-adrenergic stimulation. Notably, integrating a serines 23/24-to-alanine PKA-refractory sarcomere incorporated cTnI molecular switch complex in mice deficient in Serca2 showed blunting of ß-adrenergic stimulation-mediated enhanced diastolic heart performance. Taken together, these data provide evidence of a compensatory regulatory role of the myofilaments as a critical physiological bridging mechanism to aid in preserving heart diastolic performance in failing hearts with severe Ca2+ handling deficits.

Evaluation of abnormal gallbladder imaging findings: Surgical management and pathologic correlations in early-stage gallbladder cancer.

Gallbladder cancer is a rare but potentially fatal disease. It is often asymptomatic in early stages and is frequently found incidentally or during the workup for benign biliary disease. We present two patients who each had suspicious gallbladder imaging findings and highlight their differences on radiologic and pathologic examination.

Primary angiosarcoma of the spleen, a rare indication for splenectomy: a case report.

INTRODUCTION AND IMPORTANCE: Primary angiosarcoma of the spleen is a rare condition with a nonspecific clinical presentation and is associated with a poor prognosis. We describe two patients with primary splenic angiosarcoma successfully treated with splenectomy and adjuvant chemotherapy. CASE PRESENTATIONS: Case 1: A 50-year-old female presented with fatigue and left-sided rib, shoulder, and abdominal pain. A CT scan demonstrated a large splenic mass, and biopsy was diagnostic of angiosarcoma. An open en bloc resection of the spleen was performed, and pathologic examination confirmed high-grade angiosarcoma; the surgical margins were negative. The patient received pegylated liposomal doxorubicin (PLD) and ifosfamide; she demonstrated no evidence of recurrence with four years of follow-up. Case 2: A 70-year-old male presented with acute back pain. A CT scan demonstrated a splenic mass; biopsy was diagnostic of angiosarcoma. The patient underwent open splenectomy, and pathology revealed high-grade angiosarcoma; the surgical margins were positive. The patient received PLD and ifosfamide but presented three years later with metastatic tumor to the spine. The patient had a favorable tumor response to pembrolizumab. The patient's tumor burden remains stable at 5 years following splenectomy. CLINICAL DISCUSSION: Angiosarcoma of the spleen is a rare clinical entity and is often challenging to diagnose early. Moratality is high, especially in the case of metastasis or spontaneous rupture. CONCLUSION: Due to the rare nature of this tumor, optimal treatment is not known. Here, we show excellent response in two patients to surgery combined with adjuvant therapy.

Olfactory systems across mosquito species.

There are 3559 species of mosquitoes in the world (Harbach 2018) but, so far, only a handful of them have been a focus of olfactory neuroscience and neurobiology research. Here we discuss mosquito olfactory anatomy and function and connect these to mosquito ecology. We highlight the least well-known and thus most interesting aspects of mosquito olfactory systems and discuss promising future directions. We hope this review will encourage the insect neuroscience community to work more broadly across mosquito species instead of focusing narrowly on the main disease vectors.

Advancing physiological maturation in human induced pluripotent stem cell-derived cardiac muscle by gene editing an inducible adult troponin isoform switch.

Advancing maturation of stem cell-derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell-based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene-editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a drug-inducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate-limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC-CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult-like cardiac contractile function in hiPSC-CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca2+ transient. In comparison, isogenic unedited hiPSC-CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium-like contractile adaptations that are essential for beat-to-beat modulation of adult human heart performance. These findings have relevance to hiPSC-CM structure-function and drug-discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC-CM in cardiac regeneration/repair.

Ketorolac for Patients Undergoing Implant-Based Breast Reconstruction: Impact on Hospital Length of Stay and Postoperative Narcotic Use.

BACKGROUND: Opioid drugs have been a mainstay medication for the management of postoperative pain for several decades; however, in recent years there has been a push towards investigating alternative treatment options. Although ketorolac has been widely used by other medical and surgical specialties for analgesia, its utilization in plastic surgery has been widely debated. OBJECTIVES: The purpose of this study was to investigate the efficacy of ketorolac as an adjunct in postoperative pain management. METHODS: The authors performed a retrospective review of patients who underwent implant-based breast reconstruction after mastectomy between January 2012 and December 2016. Other risk factors, such as chronic anticoagulation, aspirin, or coagulopathies, were documented as well. RESULTS: There were 198 patients who met the inclusion criteria. The results demonstrated that patients who received ketorolac utilized significantly fewer narcotics than patients who did not: 80 mg vs 108.8 mg (P = 0.002), respectively. The results showed that patients who received ketorolac had a decreased length of hospitalization: 1.9 days vs 2.1 days (P = 0.04), respectively. CONCLUSIONS: Generous narcotic prescribing has received greater scrutiny in recent years. Aside from the risk of increased narcotic availability in the community, the side effects of nausea, puritis, and constipation delay patient recovery. These data show that patients who received ketorolac have a decreased length of hospital stay and lower narcotic use, suggesting ketorolac may be a safe and cost-effective adjustment to a multi-modal pain control regimen postoperatively.

Systematic review of spinal deformities following multi-level selective dorsal rhizotomy.

PURPOSE: Cerebral palsy is a common neurological disorder that involves spasticity of the extremities and can lead to lifelong disability. Selective dorsal rhizotomy (SDR) can improve spasticity and quality of life in these patients, but it may be associated with the development of spinal deformity. Risk factors for spinal deformity after SDR have not yet been systematically examined. METHODS: Medline, Embase, and Web of Science databases were queried for clinical studies reporting incidence of new or worsening spinal deformity, including scoliosis, after SDR. Variables that represent possible risk factors for deformity were correlated with reported incidence of deformity. RESULTS: Twenty-two articles for a total of 1485 patients met the inclusion criteria for this study. Deformity occurs among all patients with a weighted mean incidence of 28.0%. Scoliosis appears to be the most common deformity occurring with a weighted mean incidence of 31.6%. There is substantial heterogeneity between studies, limiting our analysis. Significant positive correlation was found between percent of patients that developed any type of deformity and the ratio of female to male patients, p = 0.02. Significant positive correlation was also found between percent of patients that develop scoliosis and the ratio of female to male patients, p < 0.01, and between scoliosis and the number of years to follow-up, p < 0.01. CONCLUSION: Spinal deformity is an important potential complication of SDR with scoliosis being the most common type of deformity. The major risk factor for postoperative deformity is female sex. Deformity was also found to significantly increase with extended follow-up, indicating a slow process that should be carefully monitored.

Perianal Histoplasmosis Presenting as a Mass Suspicious for Malignancy: A Case Report with Review of Gastrointestinal Manifestations of Histoplasmosis.

BACKGROUND Disseminated histoplasmosis, a disease that can present years after exposure to the causative organism, may manifest in many diverse ways. Although the gastrointestinal tract is involved in most cases, the initial presentation occurring along the gastrointestinal tract, including the colon and rectum, is infrequent. CASE REPORT This case report describes a 66-year-old male patient who presented with an indurated painful perianal lesion that appeared highly suspicious for malignancy on imaging. The patient had no known history of well-established immunocompromised state except for a short course of prednisolone for chronic obstructive pulmonary disease management. A biopsy of the mass was performed, showing chronic inflammation with clusters of epithelioid histiocytes containing characteristic, PAS-fungus stain-positive, intracellular yeast forms consistent with histoplasmosis. There was no evidence of malignancy. A subsequent work-up revealed perihilar nodularity on chest X-ray suggestive of calcified granuloma, a positive Histoplasma Capsulatum Antigen test result, and mildly decreased CD4: CD8 ratio of unknown significance. HIV testing was negative. Treatment with itraconazole and terbinafine was initiated, and at 5-months follow-up, the patient reported significant improvement in signs and symptoms, with undetectable Histoplasma antigen on repeat testing. CONCLUSIONS This case represents an extremely rare presentation of histoplasmosis infection, and highlights the fact that presenting symptoms of histoplasmosis can be vague and may mimic other disease processes, including neoplasia. Biopsy of the lesion with PAS staining and serologic testing is critical in establishing the correct diagnosis.

Minimizing transfusion in sagittal craniosynostosis surgery: the Children's Hospital of Minnesota Protocol.

PURPOSE: To assess the success of a protocol using preoperative erythropoietin (EPO) and iron with perioperative tranexamic acid (TXA) in reducing blood transfusion in sagittal craniosynostosis surgery. METHODS: A retrospective chart review of all sagittal craniosynostosis patients undergoing open repair at our institution since 2010 was conducted. A novel protocol of preoperative EPO with iron and perioperative TXA, along with a shift away from automatic transfusion, was initiated in 2014. Perioperative hemoglobin levels, length of stay, and transfusion rates were compared between the historical control and the study group receiving the protocol. RESULTS: A total of 36 patients met inclusion criteria. Twenty-eight patients were male and 8 were female. Twenty-two patients were in the control group receiving neither TXA nor EPO and automatically received a transfusion, while 14 were in the study group and received the full protocol. There were no significant demographic differences between groups. Within the control group, 100% of patients were transfused compared with 14.3% of the study group (p < 0.0001). The study group also had a shorter postoperative length of stay in the hospital (mean, 3.4 days; range, 3-6) than the control (mean, 4 days; range, 2-5.5, p = 0.038). The study group had a higher preoperative hemoglobin than the control (13.6 vs. 11.8 g/dL, p = 0.0001). CONCLUSION: Our protocol of preoperative EPO and iron with perioperative TXA increased the preoperative hemoglobin and was associated with a low transfusion rate without negatively impacting postoperative course.

Hermit crabs (Pagurus bernhardus) use visual contrast in self-assessment of camouflage.

Animals can make use of camouflage to reduce the likelihood of visual detection or recognition and thus improve their chances of survival. Background matching, where body colouration is closely matched to the surrounding substrate, is one form of camouflage. Hermit crabs have the opportunity to choose their camouflage independently of body colouration as they inhabit empty gastropod shells, making them ideal to study their choice of camouflage. We used 3D-printed artificial shells of varying contrasts against a grey substrate to test whether hermit crabs prefer shells that they perceive as less conspicuous. Contrast-minimising shells were chosen for Weber contrasts stronger than -0.5. However, in looming experiments, animals responded to contrasts as weak as -0.2, indicating that while they can detect differences between shells and the background, they are only motivated to move into those shells when the alternatives contrast strongly. This suggests a trade-off between camouflage and vulnerability introduced by switching shells.

Investigation of human iPSC-derived cardiac myocyte functional maturation by single cell traction force microscopy.

Recent advances have made it possible to readily derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). HiPSC-CMs represent a valuable new experimental model for studying human cardiac muscle physiology and disease. Many laboratories have devoted substantial effort to examining the functional properties of isolated hiPSC-CMs, but to date, force production has not been adequately characterized. Here, we utilized traction force microscopy (TFM) with micro-patterning cell printing to investigate the maximum force production of isolated single hiPSC-CMs under varied culture and assay conditions. We examined the role of length of differentiation in culture and the effects of varied extracellular calcium concentration in the culture media on the maturation of hiPSC-CMs. Results show that hiPSC-CMs developing in culture for two weeks produced significantly less force than cells cultured from one to three months, with hiPSC-CMs cultured for three months resembling the cell morphology and function of neonatal rat ventricular myocytes in terms of size, dimensions, and force production. Furthermore, hiPSC-CMs cultured long term in conditions of physiologic calcium concentrations were larger and produced more force than hiPSC-CMs cultured in standard media with sub-physiological calcium. We also examined relationships between cell morphology, substrate stiffness and force production. Results showed a significant relationship between cell area and force. Implementing directed modifications of substrate stiffness, by varying stiffness from embryonic-like to adult myocardium-like, hiPSC-CMs produced maximal forces on substrates with a lower modulus and significantly less force when assayed on increasingly stiff adult myocardium-like substrates. Calculated strain energy measurements paralleled these findings. Collectively, these findings further establish single cell TFM as a valuable approach to illuminate the quantitative physiological maturation of force in hiPSC-CMs.

Maturation status of sarcomere structure and function in human iPSC-derived cardiac myocytes.

Human heart failure due to myocardial infarction is a major health concern. The paucity of organs for transplantation limits curative approaches for the diseased and failing adult heart. Human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) have the potential to provide a long-term, viable, regenerative-medicine alternative. Significant progress has been made with regard to efficient cardiac myocyte generation from hiPSCs. However, directing hiPSC-CMs to acquire the physiological structure, gene expression profile and function akin to mature cardiac tissue remains a major obstacle. Thus, hiPSC-CMs have several hurdles to overcome before they find their way into translational medicine. In this review, we address the progress that has been made, the void in knowledge and the challenges that remain. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.

All-trans retinoic acid-triggered antimicrobial activity against Mycobacterium tuberculosis is dependent on NPC2.

A role for vitamin A in host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro studies; however, the mechanism is unclear. In this study, we demonstrate that vitamin A-triggered antimicrobial activity against M. tuberculosis requires expression of NPC2. Comparison of monocytes stimulated with all-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D3), the biologically active forms of vitamin A and vitamin D, respectively, indicates that ATRA and 1,25D3 induce mechanistically distinct antimicrobial activities. Stimulation of primary human monocytes with ATRA did not result in expression of the antimicrobial peptide cathelicidin, which is required for 1,25D3 antimicrobial activity. In contrast, ATRA triggered a reduction in the total cellular cholesterol concentration, whereas 1,25D3 did not. Blocking ATRA-induced cellular cholesterol reduction inhibits antimicrobial activity as well. Bioinformatic analysis of ATRA- and 1,25D3-induced gene profiles suggests that NPC2 is a key gene in ATRA-induced cholesterol regulation. Knockdown experiments demonstrate that ATRA-mediated decrease in total cellular cholesterol content and increase in lysosomal acidification are both dependent upon expression of NPC2. Expression of NPC2 was lower in caseous tuberculosis granulomas and M. tuberculosis-infected monocytes compared with normal lung and uninfected cells, respectively. Loss of NPC2 expression ablated ATRA-induced antimicrobial activity. Taken together, these results suggest that the vitamin A-mediated antimicrobial mechanism against M. tuberculosis requires NPC2-dependent expression and function, indicating a key role for cellular cholesterol regulation in the innate immune response.

MicroRNA-21 targets the vitamin D-dependent antimicrobial pathway in leprosy.

Leprosy provides a model to investigate mechanisms of immune regulation in humans, given that the disease forms a spectrum of clinical presentations that correlate with host immune responses. Here we identified 13 miRNAs that were differentially expressed in the lesions of subjects with progressive lepromatous (L-lep) versus the self-limited tuberculoid (T-lep) disease. Bioinformatic analysis revealed a significant enrichment of L-lep-specific miRNAs that preferentially target key immune genes downregulated in L-lep versus T-lep lesions. The most differentially expressed miRNA in L-lep lesions, hsa-mir-21, was upregulated in Mycobacterium leprae-infected monocytes. By directly downregulating Toll-like receptor 2/1 heterodimer (TLR2/1)-induced CYP27B1 and IL1B expression as well as indirectly upregulating interleukin-10 (IL-10), hsa-mir-21 inhibited expression of the genes encoding two vitamin D-dependent antimicrobial peptides, CAMP and DEFB4A. Conversely, knockdown of hsa-mir-21 in M. leprae-infected monocytes enhanced expression of CAMP and DEFB4A and restored TLR2/1-mediated antimicrobial activity against M. leprae. Therefore, the ability of M. leprae to upregulate hsa-mir-21 targets multiple genes associated with the immunologically localized disease form, providing an effective mechanism to escape from the vitamin D-dependent antimicrobial pathway.

T-cell cytokines differentially control human monocyte antimicrobial responses by regulating vitamin D metabolism.

We investigated the mechanisms by which T-cell cytokines are able to influence the Toll-like receptor (TLR)-induced, vitamin D-dependent antimicrobial pathway in human monocytes. T-cell cytokines differentially influenced TLR2/1-induced expression of the antimicrobial peptides cathelicidin and DEFB4, being up-regulated by IFN-γ, down-regulated by IL-4, and unaffected by IL-17. The Th1 cytokine IFN-γ up-regulated TLR2/1 induction of 25-hydroxyvitamin D-1α-hydroxylase (i.e., CYP27B1), leading to enhanced bioconversion of 25-hydroxyvitamin D(3) (25D(3)) to its active metabolite 1,25D(3). In contrast, the Th2 cytokine IL-4, by itself and in combination with the TLR2/1 ligand, induced catabolism of 25D(3) to the inactive metabolite 24,25D(3), and was dependent on expression of vitamin D-24-hydroxylase (i.e., CYP24A1). Therefore, the ability of T-cell cytokines to differentially control monocyte vitamin D metabolism represents a mechanism by which cell-mediated immune responses can regulate innate immune mechanisms to defend against microbial pathogens.

Convergence of IL-1beta and VDR activation pathways in human TLR2/1-induced antimicrobial responses.

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1beta and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1beta activity, involving the upregulation of both IL-1beta and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1beta was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-kappaB sites, whereas the cathelicidin promoter had three VDREs and no NF-kappaB sites. Transfection of NF-kappaB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1beta in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.