Hybrid fed-batch cultures using XCell ATF for better yield and robust clarification process

Fed-batch cell culture processes require a clarification step to isolate the protein of interest from biomass which includes whole cells, lipids and cell debris. Selecting a suitable clarification technology is critical as it can rise concerns for bioburden, pose substantial risks to protein quality, and impact subsequent downstream purification process. The current technology of depth filtration demonstrates the limitation to perform the clarification process in a sterile manner. This process also often requires additional coupling with centrifugation turning it into a multi-stage process. We report a scalable single-step novel clarification method, High Yield Harvest (HYH), for fed-batch bioreactors using the XCell ATF system. Unlike depth filtration, the HYH method allows for a sterile clarification process while maintaining cell viability to minimize protein degradation. As an added benefit, this method is shown to elevate the protein production compared to a traditional clarification process. In the case study presented, the clarification process was initiated a few days prior to the end of fed-batch cultivation using an XCell ATF system. During this period, perfusion methodologies maintained the cell viability and increased the protein production due to the replacement of nutrients and removing toxins. The same ATF system was then used to harvest the remaining protein from the bioreactor on the final day of the cultivation. These results suggest that protein production can be increased by 40% without increasing the original fed-batch process time. The entire process is performed as a single step in a sterile manner, resulting in a clarified product ready for downstream purification

Androgen Receptor CAG Repeat Polymorphism and Epigenetic Influence among the South Indian Women with Polycystic Ovary Syndrome

The present study was carried out to assess the role of androgen receptor CAG repeat polymorphism and X chromosome inactivation (XCI) pattern among Indian PCOS women and controls which has not been hitherto explored and also to test the hypothesis that shorter CAG alleles would be preferentially activated in PCOS. CAG repeat polymorphism and X chromosome methylation patterns were compared between PCOS and non-PCOS women. 250 PCOS women and 299 controls were included for this study. Androgen receptor CAG repeat sizes, XCI percentages, and clinical and biochemical parameters were measured. The mean CAG repeat number is similar between the cases (18.74±0.13) and controls (18.73±0.12). The obese PCOS women were significantly more frequent in the <18 and >20 CAG repeat category than the lean PCOS women, yielding a highly significant odds (p = 0.001). Among the women with non-random X-inactivation, alleles with <19 repeats were more frequently activated among cases than controls (p = 0.33). CAG repeat polymorphism by itself cannot be considered as a useful marker for discriminating PCOS. We observed a trend of preferential activation of the shorter allele among the PCOS cases with non random XCI pattern. In the obese PCOS women, this microsatellite variation may account for the hyperandrogenicity to a larger extent than the lean PCOS women

Histone Deacetylase Inhibition via RGFP966 Releases the Brakes on Sensory Cortical Plasticity and the Specificity of Memory Formation

Research over the past decade indicates a novel role for epigenetic mechanisms in memory formation. Of particular interest is chromatin modification by histone deacetylases (HDACs), which, in general, negatively regulate transcription. HDAC deletion or inhibition facilitates transcription during memory consolidation and enhances long-lasting forms of synaptic plasticity and long-term memory. A key open question remains: How does blocking HDAC activity lead to memory enhancements? To address this question, we tested whether a normal function of HDACs is to gate information processing during memory formation. We used a class I HDAC inhibitor, RGFP966 (C(21)H(19)FN(4)O), to test the role of HDAC inhibition for information processing in an auditory memory model of learning-induced cortical plasticity. HDAC inhibition may act beyond memory enhancement per se to instead regulate information in ways that lead to encoding more vivid sensory details into memory. Indeed, we found that RGFP966 controls memory induction for acoustic details of sound-to-reward learning. Rats treated with RGFP966 while learning to associate sound with reward had stronger memory and additional information encoded into memory for highly specific features of sounds associated with reward. Moreover, behavioral effects occurred with unusually specific plasticity in primary auditory cortex (A1). Class I HDAC inhibition appears to engage A1 plasticity that enables additional acoustic features to become encoded in memory. Thus, epigenetic mechanisms act to regulate sensory cortical plasticity, which offers an information processing mechanism for gating what and how much is encoded to produce exceptionally persistent and vivid memories. SIGNIFICANCE STATEMENT Here we provide evidence of an epigenetic mechanism for information processing. The study reveals that a class I HDAC inhibitor (Malvaez et al., 2013; Rumbaugh et al., 2015; RGFP966, chemical formula C(21)H(19)FN(4)O) alters the formation of auditory memory by enabling more acoustic information to become encoded into memory. Moreover, RGFP966 appears to affect cortical plasticity: the primary auditory cortex reorganized in a manner that was unusually “tuned-in” to the specific sound cues and acoustic features that were related to reward and subsequently remembered. We propose that HDACs control “informational capture” at a systems level for what and how much information is encoded by gating sensory cortical plasticity that underlies the sensory richness of newly formed memories

Association of CAPN10 SNPs and Haplotypes with Polycystic Ovary Syndrome among South Indian Women

Polycystic Ovary Syndrome (PCOS) is known to be characterized by metabolic disorder in which hyperinsulinemia and peripheral insulin resistance are central features. Given the physiological overlap between PCOS and type-2 diabetes (T2DM), and calpain 10 gene (CAPN10) being a strong candidate for T2DM, a number of studies have analyzed CAPN10 SNPs among PCOS women yielding contradictory results. Our study is first of its kind to investigate the association pattern of CAPN10 polymorphisms (UCSNP-44, 43, 56, 19 and 63) with PCOS among Indian women. 250 PCOS cases and 299 controls from Southern India were recruited for this study. Allele and genotype frequencies of the SNPs were determined and compared between the cases and controls. Results show significant association of UCSNP-44 genotype CC with PCOS (p = 0.007) with highly significant odds ratio when compared to TC (OR = 2.51, p = 0.003, 95% CI = 1.37–4.61) as well as TT (OR = 1.94, p = 0.016, 95% CI = 1.13–3.34). While the haplotype carrying the SNP-44 and SNP-19 variants (21121) exhibited a 2 fold increase in the risk for PCOS (OR = 2.37, p = 0.03), the haplotype containing SNP-56 and SNP-19 variants (11221) seems to have a protective role against PCOS (OR = 0.20, p = 0.004). Our results support the earlier evidence for a possible role of UCSNP-44 of the CAPN10 gene in the manifestation of PCOS

Association of vitamin D receptor gene polymorphisms with polycystic ovary syndrome among Indian women

Background & objectives: The Vitamin-D receptor (VDR) regulates vitamin D levels and calcium metabolism in the body and these are known to be associated with endocrine dysfunctions, insulin resistance and type-2 diabetes in polycystic ovarian syndrome (PCOS). Studies on VDR polymorphisms among PCOS women are sparse. We undertook this study to investigate the association pattern of VDR polymorphisms (Cdx2, Fok1, Apa1 and Taq1) with PCOS among Indian women. Methods: For the present study, 250 women with PCOS and 250 normal healthy control women were selected from Hyderabad city, Telangana, India. The four VDR polymorphisms were genotyped and analysed using ASM-PCR (allele specific multiple PCR) and PCR-RFLP (restriction fragment length polymorphism). Results: The genotype and allele frequency distributions of only Cdx2 showed significant difference between the PCOS cases and control women, indicating protective role of this SNP against PCOS phenotype. However, significant association was observed between VDR genotypes and some of the PCOS specific clinical/biochemical traits. For example, Fok1 showed a significant genotypic difference for the presence of infertility and Cdx2 genotpes showed association with testosterone levels. Further, the two haplotypes, ACCA and ACTA, were found to be significantly associated with PCOS indicating haplotype specific risk. Interpretation & conclusions: Although VDR polymorphisms have not shown significant association with PCOS, in view of functional significance of the SNPs considered, one cannot yet rule out the possibility of their association with PCOS. Further, specifically designed studies on large cohorts are required to conclusively establish the role of VDR polymorphisms in PCOS, particularly including data on vitamin D levels

Improvement in subclinical cognitive dysfunction with thyroxine therapy in hypothyroidism: A study from tertiary care center

Aim: To evaluate the effect of hypothyroidism (both overt and subclinical) on cognitive function using latencies of P300 auditory evoked potentials (AEPs). P300 latency suggests that shorter latency times are related to better cognitive performance. P300 latencies were also done after thyroxine replacement to see the effect of treatment on cognitive function. Materials and Methods: Biochemically proven new onset cases with hypothyroidism (overt and subclinical) were enrolled into the study, AEPs of these two groups when compared with matched controls. After detailed history and physical examination, P300 potentials were recorded at two points Cz and Pz (Cz: On the midline of the head at the vertex, Pz: On the midline of the head between the vertex and occipital protuberance) using a Nicolet Viking Select neuro diagnostic system version 10.0. The study was done in electrophysiology lab in Osmania Medical College. Results: A patient characteristics of both cases and controls were comparable. The cases consisted of two groups, overt hypothyroid cases 24, mean thyroid stimulating hormone (TSH) values in them was 94, subclinical cases 21 in whom mean TSH value was 12.3. Mean P300 latencies of all cases at Cz was 342.42 ± 29.5 ms, and at Pz was 345.4 ± 30 ms. Mean P300 latencies of controls at Cz was 296.4 ± 34 ms and at Pz was 297.9 ± 33 ms (difference in P < 0.001). Mean P300 values in overt cases were 362.6 ± 32.9 ms at Cz, and at Pz it was 362.5 ± 33.9 ms. Mean P300 values in subclinical cases were 319.3 ± 30.9 ms at Cz, and at Pz it was 316.4 ± 27.9 ms. P300 values in overt cases were highly significant as compared to controls, and P300 values in the subclinical cases versus controls were also significant (P < 0.001). Conclusion: P300 latency prolongation in both clinical and subclinical hypothyroid cases shows that cognitive function is affected adversely in hypothyroidism including the subclinical hypothyroid cases. Larger studies evaluating the effect of subclinical hypothyroidism on cognitive function are needed with objective means such as the AEPs P300

CAPN10 haplotype frequency distribution among PCOS cases and controls.

*<p>Standard Pearson chi square value with degree of freedom = 1.</p

Pairwise LD estimates in PCOS cases and controls. Data are D′ values.

<p>Pairwise LD estimates in PCOS cases and controls. Data are D′ values.</p

Minor allele frequencies of CAPN10 markers in different populations.

a<p>Horikawa <i>et al.</i> (2000).</p>b<p>Vollmert <i>et al.</i> (2007).</p>c<p>Evans <i>et al.</i> (2001).</p>d<p>Gonzalez <i>et al.</i> (2004).</p>e<p>Cassell <i>et al.</i> (2002).</p>f<p>Bodhini <i>et al.</i> (2010).</p>g<p>Adak <i>et al.</i> (2010).</p>h<p>Present study.</p