A novel therapeutic approach: Blocking Glioblastoma cells’ interaction with their microenvironment

Abstract Due to the highly invasive nature of Glioblastoma (GB), complete surgical resection is not feasible, while motile tumour cells are often associated with several specific brain structures that enhance treatment-resistance. Here, we investigate the therapeutic potential of Disulfiram and Carbenoxolone, that inhibit two distinct interactions between GB and the brain tissue microenvironment: stress-induced cell-matrix adhesion and gap junction mediated cell-cell communication, respectively. Increase in cell numbers of tumour-initiating cells, which are cultured in suspension as cell clusters, and adherent differentiated cells can be blocked to a similar extent by Carbenoxolone, as both cell populations form gap junctions, but the adherent differentiated cells are much more sensitive to Disulfiram treatment, which – via modulation of NF-κB signalling – interferes with cell-substrate adhesion. Interestingly, inducing adhesion in tumour-initiating cells without differentiating them does not sensitize for Disulfiram. Importantly, combining Disulfiram, Carbenoxolone and the standard chemotherapeutic drug Temozolomide reduces tumour size in an orthotopic mouse model. Isolating GB cells from their direct environment within the brain represents an important addition to current therapeutic approaches. The blockage of cellular interactions via the clinically relevant substances Disulfiram and Carbenoxolone, has distinct effects on different cell populations within a tumour, potentially reducing motility and/or resistance to apoptosis

Rosemary Reduces Heat Stress by Inducing CRYAB and HSP70 Expression in Broiler Chickens

Heat stress negatively affects poultry production and animal health. In response, animals invoke a heat stress response by inducing heat shock proteins (HSPs). Scientists are actively seeking natural products that can enhance the heat shock response. The present study aimed at assessing the effects of a purified rosemary extract comprising antioxidant compounds on the heat shock response and HSP expression profile in broiler chickens. The response of broilers to HS in the presence of purified rosemary extract was assessed using an in vivo myocardial cell model. Pathological lesions of heart tissue were examined microscopically. The levels and activities of enzymes associated with heart damage and oxidative damage were detected. Immunohistochemical staining was performed for HSPs in myocardial cells. The results showed that lactate dehydrogenase (LDH), creatine kinase (CK), and myocardial CK (CKMB) levels were reduced by the purified rosemary extract before and during heat stress. Heat stress alone increased CK and CKMB levels. The levels of oxidative damage-associated enzymes were compared between the rosemary + heat stress and heat stress-alone groups. The results indicated that in terms of these enzymes, the purified rosemary extract induced a more antioxidative state. Pathological examinations showed that heat stress caused myocardial fiber fracture, karyopyknosis, and degeneration. The addition of purified rosemary extract ameliorated these lesions to some degree, preserving more of the basic structure. Heat stress decreased the cellular levels of crystallin alpha B (CRYAB) and HSP70. The addition of the purified rosemary extract significantly increased the levels of CRYAB and HSP70 during heat stress (p<0.0001). Immunohistochemistry showed that after rosemary treatment, CRYAB and HSP70 showed more intense staining compared with the no heat stress control group. In the rosemary + heat group, after 10 hours of heat stress, the staining intensity of these two proteins remained higher than in the heat stress group. Thus, purified rosemary extract could induce high levels of HSP70 and CRYAB in chicken hearts before and during heat stress. Purified rosemary extract could be used to alleviate heat stress in broiler chickens

Molecular Epidemiological Survey of Canine Parvovirus Circulating in China from 2014 to 2019

The global distribution of canine parvovirus (CPV-2) derived from a closely related carnivore parvovirus poses a considerable threat to the dog population. The virus is continuously undergoing genetic evolution, giving rise to several variants. To investigate the prevalence of Chinese CPV-2 strains in recent years, a total of 30 CPV-2 strains were collected from 2018 to 2021 and the VP2 gene was sequenced and analyzed. Two variants, new CPV-2a (297Ala, 426Asn) and CPV-2c (426Glu), were identified. In contrast to previous reports, the CPV-2c variant has gained an epidemiological advantage over the new CPV-2a variant in China. To compensate for the relatively small sample size, 683 Chinese CPV-2 strains identified between 2014 and 2019 were retrieved from the GenBank database and previous publications, and analyses of these strains further supported our findings, which should be considered since the CPV-2c variant has been frequently associated with immune failure in adult dogs. VP2 protein sequence analysis revealed several amino acid substitutions, including Ala5Gly, Pro13Ser, Phe267Tyr, Tyr324Ile, Gln370Arg, Thr440Ala, and Lys570Arg. Phylogenetic analysis of full-length VP2 gene indicated a close relationship between Chinese strains and other Asian strains, suggesting mutual transmission between Asian countries. Furthermore, intercontinental transmission is a cause for concern. Surprisingly, two feline panleukopenia virus (FPV) strains with the Ile101Thr mutation in the VP2 protein were identified in canine fecal samples; FPV has been considered incapable of infecting dogs. This study clarified the epidemic characteristics of Chinese CPV-2 strains detected between 2014 and 2019, offering a reference for epidemic control. In addition, the detection of FPV in canine samples may provide information for future studies on the evolution of carnivore parvoviruses

Ovarian Toxicity in Female Rats after Oral Administration of Melamine or Melamine and Cyanuric Acid.

Although the toxicity of melamine to the kidneys and testes is well known, few studies have investigated the effects of melamine on female reproductive organs. Therefore, this study explores the effects of oral administration melamine or melamine and cyanuric acid for 28 days on the ovaries of female rats. Rats that were exposed to the mixture exhibited reduced ovarian and uterine weights, a shorter estrous cycle, and reduced serum estrogen and progesterone levels compared to rats that were exposed to melamine and control rats. Furthermore, morphological analysis revealed pathological changes in the ovaries of rats exposed to melamine or the mixture, such as more atretic follicles and necrosis of oocytes and granulosa cells. TUNEL staining revealed that the exposed groups had a higher proportion of TUNEL-positive granulosa cells than the control group, and the mRNA expressions of SOD1, GPX1, GPX2, P450scc, 17β-HSD I, and 17β-HSD II were reduced in the exposure groups compared with the control group. These results indicated that exposure to melamine alone or to the melamine-cyanuric acid mixture could damage the ovaries in rats

Transportation Stress and Expression of Heat Shock Protein Affecting Pork Quality

The relationship between heat shock protein (Hsp) expression and meat quality were assessed in pigs. Carcasses from 2 h- and 6 h-transported pigs had higher temperatures and lower pH and water holding capacity values in the longissimus dorsi and gluteus maximus superficialis muscles. Long journeys were associated with increased creatine kinase (CK) levels. Higher CK levels are indicative of physical stress, as the enzyme is released from muscle fibers as a result of intense muscular exertion. These physiological and enzymatic changes were correlated with increased Hsp70 and decreased Hsp90 expression levels in both skeletal muscles. Animals whose cells contained high levels of Hsp may have had an advantage due to the protective role conferred by Hsp. Reduced Hsp levels were indicative of a higher meat quality and a good welfare of the transported pigs. The stress response declined over time in response to the same stress, such as a 6-h transport stress

Goose Nephritic Astrovirus Infection of Goslings Induces Lymphocyte Apoptosis, Reticular Fiber Destruction, and CD8 T-Cell Depletion in Spleen Tissue

The emergence of a novel goose nephritic astrovirus (GNAstV) has caused economic losses to the Chinese goose industry. High viral load is found in the spleen of goslings infected with GNAstV, but pathological injuries to the spleen due to GNAstV are largely unknown. In this study, 50 two-day-old goslings were infected orally with GNAstV, and 50 goslings were treated with PBS as control. Spleens were collected at different times following infection to assess damage. GNAstV infection caused visceral gout and urate deposition in joints, and resulted in 16% mortality. GNAstV was found in the lymphocytes and macrophages within the spleen. Lymphocyte loss, especially around the white pulp, and destruction and decline in the number of reticular fibers was observed in GNAstV-infected goslings. Moreover, in GNAstV-infected goslings, ultrahistopathological examination found that splenic lymphocytes exhibited condensed chromatin and apoptotic bodies, and reticular cells displayed damage to plasma membrane integrity and swollen mitochondria. Furthermore, TUNEL staining confirmed apoptosis of lymphocytes, and the mRNA levels of Fas and FasL were significantly increased in the GNAstV-infected goslings. In addition, GNAstV infection reduced the number and protein expression of CD8. In conclusion, GNAstV infection causes lymphocyte depletion, reticular cell necrosis, reticular fiber destruction, lymphocyte apoptosis, and reduction in CD8 levels, which contribute to spleen injury

Comparative analysis of αB-crystallin expression in heat-stressed myocardial cells in vivo and in vitro.

Relationships between αB-crystallin expression patterns and pathological changes of myocardial cells after heat stress were examined in vitro and in vivo in this study using the H9C2 cell line and Sprague-Dawley rats, respectively. Histopathological lesions, characterized by acute degeneration, karyopyknosis and loss of a defined nucleus, became more severe in rat hearts over the course of heat stress treatment from 20 min to 100 min. The expression of αB-crystallin in rat hearts showed a significant decrease (P<0.05) throughout the heat stress treatment period, except at the 40 min time point. Likewise, decreased αB-crystallin expression was also observed in the H9C2 cell line exposed to a high temperature in vitro, although its expression recovered to normal levels at later time points (80 and 100 min) and the cellular damage was less severe. The results suggest that αB-crystallin is mobilized early after exposure to a high temperature to interact with damaged proteins but that the myocardial cells cannot produce sufficient αB-crystallin for protection against heat stress. Lower αB-crystallin expression levels were accompanied by obvious cell/tissue damage, suggesting that the abundance of this protein is associated with protective effects in myocardial cells in vitro and in vivo. Thus, αB-crystallin is a potential biomarker of heat stress

Localization and expression of Hsp27 and αB-crystallin in rat primary myocardial cells during heat stress in vitro.

Neonatal rat primary myocardial cells were subjected to heat stress in vitro, as a model for investigating the distribution and expression of Hsp27 and αB-crystallin. After exposure to heat stress at 42°C for different durations, the activities of enzymes expressed during cell damage increased in the supernatant of the heat-stressed myocardial cells from 10 min, and the pathological lesions were characterized by karyopyknosis and acute degeneration. Thus, cell damage was induced at the onset of heat stress. Immunofluorescence analysis showed stronger positive signals for both Hsp27 and αB-crystallin from 10 min to 240 min of exposure compared to the control cells. According to the Western blotting results, during the 480 min of heat stress, no significant variation was found in Hsp27 and αB-crystallin expression; however, significant differences were found in the induction of their corresponding mRNAs. The expression of these small heat shock proteins (sHsps) was probably delayed or overtaxed due to the rapid consumption of sHsps in myocardial cells at the onset of heat stress. Our findings indicate that Hsp27 and αB-crystallin do play a role in the response of cardiac cells to heat stress, but the details of their function remain to be investigated