Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

Enhanced protein secretion in reduced genome strains of Streptomyces lividans.

BACKGROUND: S. lividans TK24 is a popular host for the production of small molecules and the secretion of heterologous protein. Within its large genome, twenty-nine non-essential clusters direct the biosynthesis of secondary metabolites. We had previously constructed ten chassis strains, carrying deletions in various combinations of specialized metabolites biosynthetic clusters, such as those of the blue actinorhodin (act), the calcium-dependent antibiotic (cda), the undecylprodigiosin (red), the coelimycin A (cpk) and the melanin (mel) clusters, as well as the genes hrdD, encoding a non-essential sigma factor, and matAB, a locus affecting mycelial aggregation. Genome reduction was aimed at reducing carbon flow toward specialized metabolite biosynthesis to optimize the production of secreted heterologous protein. RESULTS: Two of these S. lividans TK24 derived chassis strains showed ~ 15% reduction in biomass yield, 2-fold increase of their total native secretome mass yield and enhanced abundance of several secreted proteins compared to the parental strain. RNAseq and proteomic analysis of the secretome suggested that genome reduction led to cell wall and oxidative stresses and was accompanied by the up-regulation of secretory chaperones and of secDF, a Sec-pathway component. Interestingly, the amount of the secreted heterologous proteins mRFP and mTNFα, by one of these strains, was 12 and 70% higher, respectively, than that secreted by the parental strain. CONCLUSION: The current study described a strategy to construct chassis strains with enhanced secretory abilities and proposed a model linking the deletion of specialized metabolite biosynthetic clusters to improved production of secreted heterologous proteins.

Regulation of titin-based cardiac stiffness by unfolded domain oxidation (UnDOx).

The relationship between oxidative stress and cardiac stiffness is thought to involve modifications to the giant muscle protein titin, which in turn can determine the progression of heart disease. In vitro studies have shown that S-glutathionylation and disulfide bonding of titin fragments could alter the elastic properties of titin; however, whether and where titin becomes oxidized in vivo is less certain. Here we demonstrate, using multiple models of oxidative stress in conjunction with mechanical loading, that immunoglobulin domains preferentially from the distal titin spring region become oxidized in vivo through the mechanism of unfolded domain oxidation (UnDOx). Via oxidation type-specific modification of titin, UnDOx modulates human cardiomyocyte passive force bidirectionally. UnDOx also enhances titin phosphorylation and, importantly, promotes nonconstitutive folding and aggregation of unfolded domains. We propose a mechanism whereby UnDOx enables the controlled homotypic interactions within the distal titin spring to stabilize this segment and regulate myocardial passive stiffness.

DNA Methylation Analysis Identifies Novel Epigenetic Loci in Dilated Murine Heart upon Exposure to Volume Overload.

Left ventricular (LV) dilatation, a prominent risk factor for heart failure (HF), precedes functional deterioration and is used to stratify patients at risk for arrhythmias and cardiac mortality. Aberrant DNA methylation contributes to maladaptive cardiac remodeling and HF progression following pressure overload and ischemic cardiac insults. However, no study has examined cardiac DNA methylation upon exposure to volume overload (VO) despite being relatively common among HF patients. We carried out global methylome analysis of LV harvested at a decompensated HF stage following exposure to VO induced by aortocaval shunt. VO resulted in pathological cardiac remodeling, characterized by massive LV dilatation and contractile dysfunction at 16 weeks after shunt. Although methylated DNA was not markedly altered globally, 25 differentially methylated promoter regions (DMRs) were identified in shunt vs. sham hearts (20 hypermethylated and 5 hypomethylated regions). The validated hypermethylated loci in Junctophilin-2 (Jph2), Signal peptidase complex subunit 3 (Spcs3), Vesicle-associated membrane protein-associated protein B (Vapb), and Inositol polyphosphate multikinase (Ipmk) were associated with the respective downregulated expression and were consistently observed in dilated LV early after shunt at 1 week after shunt, before functional deterioration starts to manifest. These hypermethylated loci were also detected peripherally in the blood of the shunt mice. Altogether, we have identified conserved DMRs that could be novel epigenetic biomarkers in dilated LV upon VO exposure.

Hemodynamic stress-induced cardiac remodelling is not modulated by ablation of phosphodiesterase 4D interacting protein.

Adrenergic stimulation in the heart activates the protein kinase A (PKA), which phosphorylates key proteins involved in intracellular Ca2+ handling. PKA is held in proximity to its substrates by protein scaffolds, the A kinase anchoring proteins (AKAPs). We have previously identified the transcript of phosphodiesterase 4D interacting protein (Pde4dip; also known as myomegalin), one of the sarcomeric AKAPs, as being differentially expressed following hemodynamic overload, a condition inducing hyperadrenergic state in the heart. Here, we addressed whether PDE4DIP is involved in the adverse cardiac remodelling following hemodynamic stress. Homozygous Pde4dip knockout (KO) mice, generated by CRISPR-Cas9 technology, and wild-type (WT) littermates were exposed to aortocaval shunt (shunt) or transthoracic aortic constriction (TAC) to induce hemodynamic volume overload (VO) or pressure overload (PO), respectively. The mortality, cardiac structure, function and pathological cardiac remodelling were followed up after hemodynamic injuries. The PDE4DIP protein level was markedly downregulated in volume-overloaded- but upregulated in pressure-overloaded-WT hearts. Following shunt or TAC, mortality rates were comparably increased in both genotypes. Twelve weeks after shunt or TAC, Pde4dip-KO animals showed a similar degree of cardiac hypertrophy, dilatation and dysfunction as WT mice. Cardiomyocyte hypertrophy, myocardial fibrosis, reactivation of cardiac stress genes and downregulation of ATPase, Ca2+ transporting, cardiac muscle, slow twitch 2 transcript did not differ between WT and Pde4dip-KO hearts following shunt or TAC. In summary, despite a differential expression of PDE4DIP protein in remodelled WT hearts, Pde4dip deficiency does not modulate adverse cardiac remodelling after hemodynamic VO or PO.

Heat shock protein A4 ablation leads to skeletal muscle myopathy associated with dysregulated autophagy and induced apoptosis.

BACKGROUND: Molecular chaperones assist protein folding, facilitate degradation of misfolded polypeptides, and thereby maintain protein homeostasis. Impaired chaperone activity leads to defective protein quality control that is implicated in multiple skeletal muscle diseases. The heat shock protein A4 (HSPA4) acts as a co-chaperone for HSP70. Previously, we showed that Hspa4 deletion causes impaired protein homeostasis in the heart. However, its functional role in skeletal muscle has not been explored. METHODS: We performed a comparative phenotypic and biochemical analyses of Hspa4 knockout (KO) mice with wild-type (WT) littermates. RESULTS: HSPA4 is markedly upregulated in regenerating WT muscle in vivo, and in differentiated myoblasts in vitro. Hspa4-KO mice are marked by growth retardation and increased variability in body weight, accompanied by 35% mortality rates during the peri-weaning period. The surviving Hspa4-KO mice experienced progressive skeletal muscle myopathy, characterized by increased number of muscle fibers with centralized nuclei, heterogeneous myofiber size distribution, inflammatory cell infiltrates and upregulation of embryonic and perinatal myosin heavy chain transcripts. Hspa4-KO muscles demonstrated an accumulation of autophagosome-associated proteins including microtubule associated protein1 light chain 3-II (LC3-II) and p62/sequestosome accompanied by increased number of TUNEL-positive nuclei. CONCLUSIONS: Our findings underscore the indispensable role of HSPA4 in maintenance of muscle integrity through contribution in skeletal muscle autophagy and apoptosis, which might provide a novel therapeutic strategy for skeletal muscle morbidities.

Secretome Dynamics in a Gram-Positive Bacterial Model.

Protein secretion is a central biological process in all organisms. Most studies dissecting bacterial secretion mechanisms have focused on Gram-negative cell envelopes such as that of Escherichia coli However, proteomics analyses in Gram negatives is hampered by their outer membrane. Here we studied protein secretion in the Gram-positive bacterium Streptomyces lividans TK24, in which most of the secretome is released in the growth medium. We monitored changes of the secretome as a function of growth phase and medium. We determined distinct protein classes of "house-keeping" secreted proteins that do not change their appearance or abundance in the various media and growth phases. These comprise mainly enzymes involved in cell wall maintenance and basic transport. In addition, we detected significant abundance and content changes to a sub-set of the proteome, as a function of growth in the different media. These did not depend on the media being minimal or rich. Transcriptional regulation but not changes in export machinery components can explain some of these changes. However, additional downstream mechanisms must be important for selective secretome funneling. These observations lay the foundations of using S. lividans as a model organism to study how metabolism is linked to optimal secretion and help develop rational optimization of heterologous protein production.

Neurosurgery activity levels in the United Kingdom and republic of Ireland during the first wave of the covid-19 pandemic - a retrospective cross-sectional cohort study.

The impact of Covid-19 on surgical patients worldwide has been substantial. In the United Kingdom (UK) and the Republic of Ireland (RoI), the first wave of the pandemic occurred in March 2020. The aims of this study were to: (1) evaluate the volume of neurosurgical operative activity levels, Covid-19 infection rate and mortality rate in April 2020 with a retrospective cross-sectional cohort study conducted across 16 UK and RoI neurosurgical centres, and (2) compare patient outcomes in a single institution in April-June 2020 with a comparative cohort in 2019. Across the UK and RoI, 818 patients were included. There were 594 emergency and 224 elective operations. The incidence rate of Covid-19 infection was 2.6% (21/818). The overall mortality rate in patients with a Covid-19 infection was 28.6% (6/21). In the single centre cohort analysis, an overall reduction in neurosurgical operative activity by 65% was observed between 2020 (n = 304) and 2019 (n = 868). The current and future impact on UK neurosurgical operative activity has implications for service delivery and neurosurgical training.

CRISPLD1: a novel conserved target in the transition to human heart failure.

Heart failure is a major health problem worldwide with a significant morbidity and mortality rate. Although studied extensively in animal models, data from patients at the compensated disease stage are lacking. We sampled myocardium biopsies from aortic stenosis patients with compensated hypertrophy and moderate heart failure and used transcriptomics to study the transition to failure. Sequencing and comparative analysis of analogous samples of mice with transverse aortic constriction identified 25 candidate genes with similar regulation in response to pressure overload, reflecting highly conserved molecular processes. The gene cysteine-rich secretory protein LCCL domain containing 1 (CRISPLD1) is upregulated in the transition to failure in human and mouse and its function is unknown. Homology to ion channel regulatory toxins suggests a role in Ca2+ cycling. CRISPR/Cas9-mediated loss-of-function leads to dysregulated Ca2+ handling in human-induced pluripotent stem cell-derived cardiomyocytes. The downregulation of prohypertrophic, proapoptotic and Ca2+-signaling pathways upon CRISPLD1-KO and its upregulation in the transition to failure implicates a contribution to adverse remodeling. These findings provide new pathophysiological data on Ca2+ regulation in the transition to failure and novel candidate genes with promising potential for therapeutic interventions.

Large-scale production of a thermostable Rhodothermus marinus cellulase by heterologous secretion from Streptomyces lividans.

BACKGROUND: The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. RESULTS: CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. A protocol to rapidly purify CelA to homogeneity from culture supernatants was developed and specific anti-sera raised against it. Biophysical, biochemical and immmuno-detection analyses indicate that the enzyme is intact, stable and fully functional. CelA is the most thermostable heterologous polypeptide shown to be secreted from S. lividans. CONCLUSION: This study further validates and extends the use of the S. lividans platform for production of heterologous enzymes of industrial importance and extends it to active thermostable enzymes. This study contributes to developing a platform for poly-omics analysis of protein secretion in S. lividans.

Proteomic analysis of short-term preload-induced eccentric cardiac hypertrophy.

BACKGROUND: Hemodynamic load leads to cardiac hypertrophy and heart failure. While afterload (pressure overload) induces concentric hypertrophy, elevation of preload (volume overload) yields eccentric hypertrophy and is associated with a better outcome. Here we analysed the proteomic pattern of mice subjected to short-term preload. METHODS AND RESULTS: Female FVB/N mice were subjected to aortocaval shunt-induced volume overload that leads to an eccentric hypertrophy (left ventricular weight/tibia length +31 %) with sustained systolic heart function at 1 week after operation. Two-dimensional gel electrophoresis (2-DE) followed by mass spectrometric analysis showed alteration in the expression of 25 protein spots representing 21 different proteins. 64 % of these protein spots were up-regulated and 36 % of the protein spots were consistently down-regulated. Interestingly, α-1-antitrypsin was down-regulated, indicating higher elastin degradation and possibly contributing to the early dilatation. In addition to contractile and mitochondrial proteins, polymerase I and transcript release factor protein (PTRF) was also up-regulated, possibly contributing to the preload-induced signal transduction. CONCLUSIONS: Our findings reveal the proteomic changes of early-stage eccentric myocardial remodeling after volume overload. Induced expression of some of the respiratory chain enzymes suggests a metabolic shift towards an oxidative phosphorylation that might contribute to the favorable remodeling seen in early VO. Down-regulation of α-1-antitrypsin might contribute to extracellular matrix remodeling and left ventricular dilatation. We also identified PTRF as a potential signaling regulator of volume overload-induced cardiac hypertrophy.

Respiratory distress and early neonatal lethality in Hspa4l/Hspa4 double-mutant mice.

Heat shock proteins HSPA4L and HSPA4 are closely related members of the HSP110 family and act as cochaperones. We generated Hspa4l(-/-)Hspa4(-/-) mice to investigate a functional complementarity between HSPA4L and HSPA4 during embryonic development. Hspa4l(-/-)Hspa4(-/-) embryos exhibited marked pulmonary hypoplasia and neonatal death. Compared with lungs of wild-type, Hspa4l(-/-), and Hspa4(-/-) embryos, Hspa4l(-/-)Hspa4(-/-) lungs were characterized by diminished saccular spaces and increased mesenchymal septa. Mesenchymal hypercellularity was determined to be due to an increased cell proliferation index and decreased cell death. A significant increase in expression levels of prosurvival protein B cell leukemia/lymphoma 2 may be the cause for inhibition of apoptotic process in lungs of Hspa4(-/-)Hspa4l(-/-) embryos. Accumulation of glycogen and diminished expression of surfactant protein B, prosurfactant protein C, and aquaporin 5 in saccular epithelium suggested impaired maturation of type II and type I pneumocytes in the Hspa4l(-/-)Hspa4(-/-) lungs. Further experiments showed a significant accumulation of ubiquitinated proteins in the lungs of Hspa4l(-/-)Hspa4(-/-) embryos, indicating an impaired chaperone activity. Our study demonstrates that HSPA4L and HSPA4 collaborate in embryonic lung maturation, which is necessary for adaptation to air breathing at birth.

A Single-Centre Experience of the Management and Surgical Outcomes of Late-Onset Idiopathic Aqueductal Stenosis.

Background Although idiopathic aqueductal stenosis is a congenital disorder, some patients present in adulthood. Many theories have tried to account for the late-onset presentation; however, the aetiology remains uncertain. This study aimed to investigate the clinical presentation, management, and outcomes of patients with late-onset idiopathic aqueductal stenosis (LIAS) managed at our centre. Methodology A retrospective study of patients with a diagnosis of LIAS managed at our centre between 1996 and 2018 was undertaken. Data on clinical presentation, imaging, management, and outcomes were retrieved from patient records and radiology reports. Results A total of 20 patients were diagnosed with LIAS during the study period. Endoscopic third ventriculostomy (ETV) was the initial modality of treatment for nine patients, ventriculoperitoneal shunt (VPS) for four patients, and conservative management in seven patients, in four of them intracranial pressure (ICP) was found to be normal following a period of ICP monitoring. The median follow-up period was three years (1 month to 24 years). One patient was lost to follow-up. One ETV failed in the first six months necessitating VPS insertion. Two cases that were initially managed conservatively required a VPS three and nine years following the initial presentation. Of the patients undergoing VPS insertion, all subsequently required valve adjustment or surgical revision. Conclusions The majority of patients with LIAS undergoing ETV were managed successfully, whereas VPS insertion was associated with a high rate of revision surgery in this cohort. ETV should be considered as the treatment of choice to avoid the long-term complications of shunting for patients with LIAS.

COVID-19 and Spontaneous Resolution of Lumbar Disk Prolapse: A Retrospective Cohort Study of Patients Awaiting Microdiscectomy.

BACKGROUND: Between individual patients with lumbar disk prolapse (LDP), the natural course of disease is significantly variable. Spontaneous resolution is reported to occur in up to 70% of cases. However, we currently cannot predict for whom and when this will occur. Neurosurgical intervention is indicated for LDP patients with nontolerable pain after at least 8 to 12 weeks of conservative management, or significant neurologic deficit. Channeling essential resources in the National Health Service (NHS) to fight the COVID-19 pandemic led to the postponement of most elective operations, including microdiskectomy. This left many LDP patients previously considered to be surgical candidates with conservative-only options in the interim. To our knowledge, we are the first center to report the specific impact of the peri- and postpandemic period on waiting list times, delayed elective microdiskectomy, and the incidence of spontaneous LDP resolution. METHODS: Retrospective case series of a prospectively collected electronic departmental database identified LDP patients who would have been impacted by the COVID-19 pandemic at some point in their care pathway (March 2020-February 2022). Further information was obtained from electronic patient records. RESULTS: In total, 139 LDP patients were listed for elective microdiskectomy at the time of postponement of elective surgery. Over a third of LDP patients (n = 47, 33.8%), in shared decision with the responsible neurosurgeon, had their rescheduled microdiskectomy canceled due to clinical improvement (14.1%), radiologic regression (6.5%), or both (12.2%). CONCLUSION: Our single-center retrospective analysis revealed that for over a third of LDP patients, the prolonged postpandemic waiting list times for elective microdiskectomy resulted in their surgery not taking place either due to spontaneous clinical improvement or proven radiologic regression. Considering this, a prolonged conservative approach to LDP may be appropriate in some patients, allowing time for natural resolution, while avoiding perioperative risks.

Apo and Aß46-bound γ-secretase structures provide insights into amyloid-ß processing by the APH-1B isoform.

Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.

Dundee Annual Neurosurgery Skills Event (DANSE)-Improving the Availability and Affordability of Neurosurgical Skills Workshops for Medical Students.

Background Neurosurgery can be a daunting career choice for medical students, with preparation for trainee application often being inaccessible and expensive. This article describes a student-led neurosurgical skills event supported by local neurosurgery faculty members. Such event was designed to offer a means to bridge this gap by providing an opportunity to practice neurosurgical techniques in simulation, and learn about what a career in neurosurgery involves. Methods Pre- and postskills laboratory surveys were used to ascertain the baseline confidence and knowledge of common neurosurgical techniques, as well as to what both the application to neurosurgery and the typical workload of a neurosurgeon involves. The conference offered six neurosurgical workshops as well as three lectures to provide practical and theoretical learning opportunities. The session included introduction to the candidates and faculty, identification of learning objectives, and career discussion. Postcourse feedback also was also used to assess learning outcomes. Results Eighteen students attended the event. Postskills event, students were significantly more likely to understand the principles behind all of the relevant neurosurgical skills included on the day. Additionally, students were more likely to understand what a career in neurosurgery involves, and how to approach applying for a training number. Respondents enjoyed the workshops, valued hands-on experience and interactions with consultants, found it affordable, and would recommend to their peers. Conclusions For medical students interested in a career in neurosurgery, opportunities to learn relevant techniques and skills are often expensive and difficult to come across. Here, we highlight affordable methods of simulation to result in significant student satisfaction. Additionally, providing ample opportunity to practice different neurosurgical techniques under almost 1:1 level tutoring enables significant increases in students' confidence and understanding of different neurosurgical concepts. We greatly encourage other medical student groups to develop their own hands-on simulation events to attract medical students to a surgical field often considered daunting and inaccessible, and address gaps in the medical school curriculum.

A suspension of inactivated bacteria used for vaccination against recurrent urinary tract infections increases the phagocytic activity of murine macrophages.

Background: Urinary tract infections are a major cause of the consumption of antibiotics in humans. Methods: We studied the effect of a vaccine (StroVac®, containing inactivated bacteria and used to prevent recurrent urinary tract infections) licensed in Germany on the release of pro-inflammatory cytokines and the phagocytosis of Escherichia (E.) coli in primary murine macrophages and the macrophage cell line J774A.1. Results: StroVac® increased the release of the cytokines TNF-α, IL-6, IL-12/23 p40, and IL-1ß and stimulated the phagocytosis of E. coli in a dose-dependent manner. This effect was independent of LPS as shown by the use of macrophages isolated from LPS-resistant C3H/HeJ mice. At concentrations up to 30 mg/l it was not toxic to bacteria or eukaryotic cells. Conclusion: StroVac® does not only act via the adaptive but also by stimulating the innate immune system. This stimulation may help to build trained innate immunity against bacterial pathogens involved in recurrent urinary tract infections.

Novel bis-amide-based bis-thiazoles as Anti-colorectal Cancer Agents Through Bcl-2 Inhibition: Synthesis, In Vitro, and In Vivo studies.

BACKGROUND: Some heterocycles having bisamide linkage are receiving much interest due to their remarkable biological potencies and they are naturally occurring. Some bisamides and thiazole derivatives were found to inhibit the protein levels of Bcl-2 significantly. This prompted us to synthesize new bis(heterocyclic) derivatives having bisamide function to explore their anti-cancer activities. METHODS: Novel bis-amide-based bis-thiazoles and thiadiazoles were synthesized by reaction of a new bisthiosemicarbazone with a variety of hydrazonoyl chlorides, a-chloroacetylacetone and haloacetic acid derivatives. Most of the synthesized derivatives were tested for colorectal (HCT-116) and breast (MCF-7) cell lines using the MTT assay, with the apoptotic investigation through flow cytometric and RT-PCR analyses. RESULTS: Some derivatives were found to be highly cytotoxic against HCT-116 cells with an IC50 range of (10.44-13.76 µM) compared to 5-fluorouracil (5-FU) (IC50 = 11.78 µM). One product significantly stimulated apoptotic colorectal cancer cell death by 27.24-fold (50.13% compared to control 1.84%) by arresting the cell cycle at the G2/M phase. The obtained results revealed that compound 7f was more cytotoxic against HCT-116 cells than 5-FU. Compound 7f remarkably enhanced apoptotic colorectal cancer cell death and upregulated the propapoptotic genes (P53, BAX and Capases-3,-8,-9) and downregulated the anti-apoptotic gene, B-cell lymphoma 2 (Bcl-2). In vivo study exhibited that 7f-treatment caused tumor inhibition ratio (TIR%) of 50.45% compared to 54.86% in the 5-FU treatment, with a significant reduction in tumor mass and volume. The anti-tumor activity of compound 7f was accompanied by ameliorated hematological and biochemical analyses, histopathological improvement in treated liver tissues, and the immunohistochemical staining revealed Bcl-2 inhibition in agreement with the in vitro results. CONCLUSION: Compound 7f is an interesting candidate for further development as a chemotherapeutic anti-cancer agent.

Metabolomic Profiling in Patients with Different Hemodynamic Subtypes of Severe Aortic Valve Stenosis.

Severe aortic stenosis (AS) is a common pathological condition in an ageing population imposing significant morbidity and mortality. Based on distinct hemodynamic features, i.e., ejection fraction (EF), transvalvular gradient and stroke volume, four different AS subtypes can be distinguished: (i) normal EF and high gradient, (ii) reduced EF and high gradient, (iii) reduced EF and low gradient, and (iv) normal EF and low gradient. These subtypes differ with respect to pathophysiological mechanisms, cardiac remodeling, and prognosis. However, little is known about metabolic changes in these different hemodynamic conditions of AS. Thus, we carried out metabolomic analyses in serum samples of 40 AS patients (n = 10 per subtype) and 10 healthy blood donors (controls) using ultrahigh-performance liquid chromatography-tandem mass spectroscopy. A total of 1293 biochemicals could be identified. Principal component analysis revealed different metabolic profiles in all of the subgroups of AS (All-AS) vs. controls. Out of the determined biochemicals, 48% (n = 620) were altered in All-AS vs. controls (p < 0.05). In this regard, levels of various acylcarnitines (e.g., myristoylcarnitine, fold-change 1.85, p < 0.05), ketone bodies (e.g., 3-hydroxybutyrate, fold-change 11.14, p < 0.05) as well as sugar metabolites (e.g., glucose, fold-change 1.22, p < 0.05) were predominantly increased, whereas amino acids (e.g., leucine, fold-change 0.8, p < 0.05) were mainly reduced in All-AS. Interestingly, these changes appeared to be consistent amongst all AS subtypes. Distinct differences between AS subtypes were found for metabolites belonging to hemoglobin metabolism, diacylglycerols, and dihydrosphingomyelins. These findings indicate that relevant changes in substrate utilization appear to be consistent for different hemodynamic subtypes of AS and may therefore reflect common mechanisms during AS-induced heart failure. Additionally, distinct metabolites could be identified to significantly differ between certain AS subtypes. Future studies need to define their pathophysiological implications.

Targeted disruption of Hspa4 gene leads to cardiac hypertrophy and fibrosis.

Failure of molecular chaperones to direct the correct folding of newly synthesized proteins leads to the accumulation of misfolded proteins in cells. HSPA4 is a member of the heat shock protein 110 family (HSP110) that acts as a nucleotide exchange factor of HSP70 chaperones. We found that the expression of HSPA4 is upregulated in murine hearts subjected to pressure overload and in failing human hearts. To investigate the cardiac function of HSPA4, Hspa4 knockout (KO) mice were generated and exhibited cardiac hypertrophy and fibrosis. Hspa4 KO hearts were characterized by a significant increase in heart weight/body weight ratio, elevated expression of hypertrophic and fibrotic gene markers, and concentric hypertrophy with preserved contractile function. In response to pressure overload, cardiac hypertrophy and remodeling were further aggravated in the Hspa4 KO compared to wild type (WT) mice. Cardiac hypertrophy in Hspa4 KO hearts was associated with enhanced activation of gp130-STAT3, CaMKII, and calcineurin-NFAT signaling. Protein blot and immunofluorescent analyses showed a significant accumulation of polyubiquitinated proteins in cardiac cells of Hspa4 KO mice. These results suggest that the myocardial remodeling of Hspa4 KO mice is due to accumulation of misfolded proteins resulting from impaired chaperone activity. Further analyses revealed a significant increase in cross sectional area of cardiomyocytes, and in expression levels of hypertrophic markers in cultured neonatal Hspa4 KO cardiomyocytes suggesting that the hypertrophy of mutant mice was a result of primary defects in cardiomyocytes. Gene expression profile in hearts of 3.5-week-old mice revealed a differentially expressed gene sets related to ion channels, muscle-specific contractile proteins and stress response. Taken together, our in vivo data demonstrate that Hspa4 gene ablation results in cardiac hypertrophy and fibrosis, possibly, through its role in protein quality control mechanism.