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Background The cell suicide pathway of apoptosis is a necessary event in the life of multicellular organisms. It is involved in many biological processes ranging from development to the immune response. Evolutionarily conserved proteases, called caspases, play a central role in regulating apoptosis. Reception of death stimuli triggers the activation of initiator caspases, which in turn activate the effector caspases. In Lepidoptera, apoptosis is crucial in processes such as metamorphosis or defending against baculovirus infection. The discovery of p35, a baculovirus protein inhibiting caspase activity, has led to the characterization of the first lepidopteran caspase, Sf-Caspase-1. Studies on Sf-Caspase-1 mode of activation suggested that apoptosis in Lepidoptera requires a cascade of caspase activation, as demonstrated in many other species. Results In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived EST datasets. We identified 66 sequences distributed among 27 species encoding putative caspases. Phylogenetic analyses showed that Lepidoptera possess at least 5 caspases, for which we propose a unified nomenclature. According to homology to their Drosophila counterparts and their primary structure, we determined that Lep-Caspase-1, -2 and -3 are putative effector caspases, whereas Lep-Caspase-5 and -6 are putative initiators. The likely function of Lep-Caspase-4 remains unclear. Lep-Caspase-2 is absent from the silkworm genome and appears to be noctuid-specific, and to have arisen from a tandem duplication of the Caspase-1 gene. In the tobacco hawkmoth, 3 distinct transcripts encoding putative Caspase-4 were identified, suggesting at least 2 duplication events in this species. Conclusions The basic repertoire of five major types of caspases shared among Lepidoptera seems to be smaller than for most other groups studied to date, but gene duplication still plays a role in lineage-specific increases in diversity, just as in Diptera and mammals.

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival ( P  = 0.008 and P  = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086–1.670; P  = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups ( P  = 0.001) and in non-basal like subgroup (P  = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort ( P  = 0.005) and basal-like subgroup ( P  = 0.034), as indicated by Kaplan–Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts.

The prognostic value of proliferation index (PI) and apoptotic index (AI), caspase-8, -9 and -10 expression have been investigated in primary Ewing's sarcoma family of tumours (ESFT). Proliferating cells, detected by immunohistochemistry for Ki-67, were identified in 91% (91/100) of tumours with a median PI of 14 (range 0-87). Apoptotic cells, identified using the TUNEL assay, were detected in 96% (76/79) of ESFT; the median AI was 3 (range 0-33). Caspase-8 protein expression was negative (0) in 14% (11/79), low (1) in 33% (26/79), medium (2) in 38% (30/79) and high (3) in 15% (12/79) of tumours, caspase-9 expression was low (1) in 66% (39/59) and high (3) in 34% (20/59), and caspase-10 protein was low (1) in 37% (23/62) and negative (0) in 63% (39/62) of primary ESFT. There was no apparent relationship between caspase-8, -9 and -10 expression, PI and AI. PI was predictive of relapse-free survival (RFS; p = 0.011) and overall survival (OS; p = <0.001) in a continuous model, whereas AI did not predict outcome. Patients with tumours expressing low levels of caspase-9 protein had a trend towards a worse RFS than patients with tumours expressing higher levels of caspase-9 protein (p = 0.054, log rank test), although expression of caspases-8, -9 and/or -10 did not significantly predict RFS or OS. In a multivariate analysis model that included tumour site, tumour volume, the presence of metastatic disease at diagnosis, PI and AI, PI independently predicts OS (p = 0.003). Consistent with previous publications, patients with pelvic tumours had a significantly worse OS than patients with tumours at other sites (p = 0.028); patients with a pelvic tumour and a PI≥20 had a 6 fold-increased risk of death. These studies advocate the evaluation of PI in a risk model of outcome for patients with ESFT.

This protocol describes HyCoSuL, an approach that uses tetrapeptides containing natural and >100 unnatural amino acids to screen for protease substrate specificity and to engineer highly active and selective substrates and activity-based probes. Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids. HyCoSuL is a combinatorial library of tetrapeptides containing amino acid mixtures at the P4–P2 positions, a fixed amino acid at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1′ position. Once the peptide is recognized and cleaved by a protease, the ACC is released and produces a readable fluorescence signal. Here, we describe the synthesis and screening of HyCoSuL for human caspases and legumain. We also discuss possible modifications and adaptations of this approach that make it a useful tool for developing highly active and selective reagents for a wide variety of proteolytic enzymes. The protocol can be divided into three major parts: (i) solid-phase synthesis of the fluorescence-labeled HyCoSuL, (ii) screening of protease P4–P2 preferences, and (iii) synthesis of the optimized activity probes equipped with an AOMK (acyloxymethyl ketone) reactive group and a biotin label for easy detection. Beginning with the library design, the entire protocol can be completed in 4–8 weeks (HyCoSuL synthesis: 3–5 weeks; HyCoSuL screening per enzyme: 4–8 d; and activity-based probe synthesis: 1–2 weeks).

Background Submental fullness is perceived as unattractive by both men and women. The noninvasive simultaneous delivery of HIFES and synchronized radiofrequency+ (Sync RF+) technologies aims to address the submental fullness by concurrently targeting the skin, adipose tissue, and weakened anterior belly of the digastric muscle, the three contributing layers to the double chin appearance. Aims This study aims to investigate the histological changes to adipose tissue related to cell morphology, caspase‐7, and Bcl‐2 levels to detect adipocyte apoptosis following the HIFES and Sync RF+ treatment on human subjects. Methods The active group (n = 6) received single 20‐min treatment on the submental area, while the control group (n = 2) did not receive any treatment. Biopsies of subcutaneous fat tissue were obtained at baseline, and 24 h and 7 days posttreatment. The specimens were histologically and immunohistochemically analyzed for changes in morphology, caspase‐7, and Bcl‐2 levels. Results Observed caspase‐7 levels increased by 511% at 24‐h posttreatment, and 101% at 7 days (p < 0.0001), while the Bcl‐2 levels decreased by 89% at 24 h and 24% at 7 days posttreatment (p < 0.0001). The control group had no statistically significant relative changes in the activity of caspase‐7. Posttreatment adipocytes were shrunken in size, and shapes lost their uniformity compared to baseline. Five of six subjects reported the treatment as being comfortable. No adverse events were observed during the study. Conclusions The results of this human histology study indicate that noninvasive HIFES and Sync RF+ technologies have a favorable safety profile for submental fat reduction through the induction of adipocyte apoptosis. Trial Registration ClinicalTrials.gov identifier: NCT06282172

The pathogenesis of Alzheimer’s disease (AD) remains to be elucidated. Oxidative damage and excessive beta-amyloid oligomers are components of disease progression but it is unclear how these factors are temporally related. At post mortem, the superior temporal gyrus (STG) of AD cases contains plaques, but displays few tangles and only moderate neuronal loss. The STG at post mortem may represent a brain region that is in the early stages of AD or alternately a region resistant to AD pathogenesis. We evaluated expression profiles and activity of endogenous anti-oxidants, oxidative damage and caspase activity in the STG of apolipoprotein ε4-matched human AD cases and controls. Total superoxide dismutase (SOD) activity was increased, whereas total glutathione peroxidase (GPX), catalase (CAT) and peroxiredoxin (Prx) activities, were decreased in the AD-STG, suggesting that hydrogen peroxide accumulates in this brain region. Transcripts of the transcription factor NFE2L2 and inducible HMOX1 , were also increased in the AD-STG, and this corresponded to increased Nuclear factor erythroid 2-related factor (NRF-2) and total heme-oxygenase (HO) activity. The protein oxidation marker 4-hydroxynonenal (4-HNE), remained unchanged in the AD-STG. Similarly, caspase activity was unaltered, suggesting that subtle redox imbalances in early to moderate stages of AD do not impact STG viability.

Background and Objectives: Green tea is known for its powerful antioxidant properties. However, the effects of green tea consumption during pregnancy on neonatal development and the mechanisms of these effects are not fully understood. The aim of this study was to investigate potential damage to atrial cardiomyocytes of newborn rat pups whose mothers received green tea during pregnancy and to elucidate the apoptotic mechanisms underlying this possible damage. Materials and Methods: Wistar albino rats (weighing 200–220 g, 10 weeks old) were used in this study. Following the confirmation of pregnancy, rats were randomly assigned to groups, and the experimental group was administered green tea by oral gavage at a dose of 50 mg/kg per day for 21 days. Atrial cardiomyocytes and mitral valve cells from newborn pups (postnatal day 1) were obtained and evaluated immunohistochemically for cytochrome c, caspase-9, and caspase-3 expression. Results: TUNEL analysis revealed a significant increase in DNA fragmentation in the green tea group, with the median number of apoptotic cells per region of interest (ROI) rising from 5.5 to 24.5 in atrial cardiomyocytes (p < 0.001), and from 2.0 to 10.0 in mitral valve cells (p < 0.05). Immunohistochemically, the control group showed faint-to-weak basal immunoreactivity of cytochrome c and caspase-3, and weak-to-moderate expression of caspase-9. In the green tea group, caspase-3 immunoreactivity was moderate, while cytochrome c and caspase-9 immunoreactivity were significantly higher. Quantitative HSCORE analysis confirmed significant elevations in atrial cardiomyocytes for cytochrome c (from 65.0 to 210.0; p < 0.001), caspase-9 (from 85.0 to 140.0; p < 0.001), and caspase-3 (from 60.0 to 120.5; p < 0.001). Similar statistically significant increases were observed across all corresponding markers in the mitral valve cells (p < 0.05). Overall, the induction of apoptosis was notably more pronounced in atrial cardiomyocytes than in mitral valve cells. Conclusions: Our findings suggest that the mechanism of potential damage in atrial cardiomyocytes of newborn rat pups is associated with mitochondria-mediated apoptosis, potentially triggered by activation of the cytochrome c, caspase-9 and caspase-3 axis. These results highlight the importance of exercising caution regarding the consumption of green tea supplements during pregnancy. Further studies are needed to correlate these preliminary neonatal observations with clinical outcomes.

BACKGROUND: Locoweeds cause significant livestock poisoning and economic loss all over the world. Animals can develop locoism, a chronic neurological disease, after grazing on locoweeds. Oxytropis kansuensis is a variety of locoweed that contains swainsonine as its main toxic ingredient. The purpose of this study was to investigate the apoptotic pathway induced in the cerebrum by swainsonine. RESULTS: Twenty-four Sprague-Dawley rats were randomly divided into four groups (experimental groups I, II, III and a control group) and 6 SD rats of each group were feed in 3 cages separately. Rats were penned as groups and fed with feeds containing 15% (SW content 0.03‰), 30% (SW content 0.06‰), or 45% (SW content 0.09‰) O. kansuensis for experimental groups I, II, and III, respectively, or complete feed in the case of the control group. One hundred and nineteen days after poisoning, and all rats showed neurological disorders at different degrees, which were considered to be successful established a chronic poisoning model of O. kansuensis. rats were sacrificed and the expression of Fas, FasL, Bcl-2, Bax as well as cleaved caspase-3, -8 and -9 proteins in brain tissues were detected by Western blot. The results showed that SW treatment up-regulated Fas and Fas ligand (FasL) (P < 0.05), and that there was an increase in Bax and a decrease in Bcl-2 protein (P < 0.01). Moreover, SW treatment significantly increases the activation of caspase-3, 8 and -9, the key effectors in apoptosis pathway (P < 0.01). CONCLUSION: Our data suggest that SW induces apoptosis in cells of the brain through death receptor and mitochondria-mediated, caspase-dependent apoptotic pathways in the brain tissue of SD rats.

In this study, we used proteomics and biological network analysis to evaluate the potential biological processes and components present in the identified proteins of biopsies from cutaneous leishmaniasis (CL) patients infected by Leishmania braziliensis in comparison with normal skin. We identified 59 proteins differently expressed in samples from infected and normal skin. Biological network analysis employing identified proteins showed the presence of networks that may be involved in the cell death mediated by cytotoxic T lymphocytes. After immunohistochemical analyses, the expression of caspase-9, caspase-3, and granzyme B was validated in the tissue and positively correlated with the lesion size in CL patients. In conclusion, this work identified differentially expressed proteins in the inflammatory site of CL, revealed enhanced expression of caspase-9, and highlighted mechanisms associated with the progression of tissue damage observed in lesions.

Background We previously described a sub-population of epithelial ovarian cancer (EOC) cells with a functional TLR-4/MyD88/NF-κB pathway (Type I EOC cells), which confers the capacity to respond to Paclitaxel, a known TLR-4 ligand, by enhancing NF-κB activity and upregulating cytokine secretion – events that are known to promote tumor progression. It is therefore important to distinguish those patients that should not receive Paclitaxel; it is also important to identify alternative chemotherapy options that would benefit this sub-group of patients. The objective of this study is to determine if the KSP inhibitor, ARRY-520, can be a substitute for Paclitaxel in patients with Type I EOC. Methods EOC cells isolated from either ascites or tumor tissue were treated with increasing concentrations of ARRY-520 or Paclitaxel and cell viability determined. Activation of the apoptotic pathway was determined using Western blot analysis. Mitochondrial integrity was quantified using JC1 dye. Cytokine profiling was performed from supernatants using xMAP technology. NF-κB activity was measured using a Luciferase reporter system. In vivo activity was determined using a subcutaneous xenograft mouse model. Results ARRY-520 and Paclitaxel exhibited the same cytotoxic effect on Type I and II cells. The GI 50 at 48 h for Type II EOC cells was 0.0015 μM and 0.2 μM for ARRY-520 and Paclitaxel, respectively. For Type I EOC cells, the GI 50 at 48 h was > 3 μM and >20 μM for ARRY-520 and Paclitaxel, respectively. Decrease in the number of viable cells was accompanied by mitochondrial depolarization and caspase activation. Unlike Paclitaxel, ARRY-520 did not induce NF-κB activation, did not enhance cytokine secretion, nor induce ERK phosphorylation in Type I EOC cells. Conclusion Administration of Paclitaxel to patients with high percentage Type I cancer cells could have detrimental effects due to Paclitaxel-induced enhancement of NF-κB and ERK activities, and cytokine production (e.g. IL-6), which promote chemoresistance and tumor progression. ARRY-520 has similar anti-tumor activity in EOC cells as that of Paclitaxel. However, unlike Paclitaxel, it does not induce these pro-tumor effects in Type I cells. Therefore, the KSP inhibitor ARRY-520 may represent an alternative to Paclitaxel in this subgroup of EOC patients.