Quality-by-Design: Are We There Yet?

In 2012, the Quality-by-Design and Product Performance Focus Group of AAPS conducted a survey to assess the state of adoption and perception of Quality-by-Design (QbD). Responses from 149 anonymous individuals from industry—including consultants—(88%), academia (7%), and regulatory body (4%), were collected. A majority of respondents (54% to 76%) reported high frequency of utilization of several tools and most QbD elements outlined by International Conference on Harmonization Q8, with design of experiments, risk assessment, and the quality target product profile ranked as the top three. Over two thirds of respondents agreed that the benefits of QbD included both the positive impact it can have on the patient (78%), as well as on internal processes such as knowledge management (85%), decision making (79%), and lean manufacture (71%). However, more than 50% from industry were neutral about or disagreed with QbD leading to a better return on investment. This suggests that, despite the recognized scientific, manufacture, and patient-related benefits, there is not yet a clearly articulated business case for QbD available. There was a difference of opinion between industry and regulatory agency respondents as to whether a QbD-based submission resulted in increased efficiency of review. These contrasting views reinforce the idea that QbD implementation can benefit from further dialog between industry and regulatory authorities. A majority of respondents from academia indicated that QbD has influenced their research. In total, the results indicate the broad adoption of QbD but also suggest we are yet in a journey and that the process of gathering all experience and metrics required for connecting and demonstrating QbD benefits to all stakeholders is still in progress.     

c-KIT positive Gastrointestinal Stromal Tumor presenting with acute bleeding in a patient with neurofibromatosis type 1: a case report

Background

Gastrointestinal stromal tumours are rare (GIST). However, the incidence of GIST among neurofibromatosis type 1 (NF-1) patients is approximately 3.9-25%. GIST can present clinically in different ways such as abdominal pain, gastrointestinal bleeding and obstruction.

Case report

We present 51 year female patient admitted with Background of neurofibromatosis type 1 admitted with melena. OGD has been done and showed duodenitis with large volume fresh blood in distal duodenum but no obvious bleeding point. Exploratory laparotomy revealed smooth nodular masses on the serosal surface of jejunum. Small bowel resection and side-to-side anastomosis were performed. Histopathoogical examination revealed small bowel gasrointestinal stromal tumour with low risk malignant potential.

Conclusion

The incidence of GIST among neurofibromatosis type 1 (NF-1) patients is not uncommon and we should pay attention to gastrointestinal manifestation in such patients.

     

Immunization with recombinant adenovirus synthesizing the secretory form of Japanese encephalitis virus envelope protein protects adenovirus-exposed mice against lethal encephalitis

Replication-defective recombinant adenoviruses (RAds) were constructed that synthesized the pre-membrane and envelope (E) proteins of Japanese encephalitis virus (JEV). Recombinant virus RAdEa synthesized Ea, the membrane-anchored E protein, and RAdEs synthesized Es, the secretory E protein. Compared with RAdEs, RAdEa replicated poorly in HEK 293A cells and synthesized lower amounts of E protein. Oral immunization of mice with RAds generated low titers of anti-JEV antibodies that had little JEV neutralizing activity. Intra-muscular (IM) immunization of mice with either RAd generated high titers of anti-JEV antibodies. Interestingly, RAdEa induced only low titers of JEV neutralizing antibodies. Titers were significantly higher in case of RAdEs immunization. Splenocytes from mice immunized IM with RAds secreted large amounts of interferon-γ and moderate amounts of interleukin-5 in the presence of JEV and showed cytotoxic activity against JEV-infected cells. Naïve mice immunized IM with RAdEs showed complete protection against a lethal dose of JEV given intra-cerebrally. In order to study the effect of the pre-existing adenovirus 5 (Ad5) immunity on the outcome of the RAdEs immunization, mice were exposed to Ad5 through IM or intra-nasal (IN) routes before immunization with RAdEs. Mice exposed to Ad5 through the IN route, when immunized with RAdEs given IM, or those exposed to Ad5 through the IM route, when immunized with RAdEs given IN, were completely protected against lethal JEV challenge.     

Sterol glycosyltransferases required for adaptation of Withania somnifera at high temperature

Heat is a major environmental stress factor that confines growth, productivity, and metabolism of plants. Plants respond to such unfavorable conditions through changes in their physiological, biochemical and developmental processes. Withania somnifera, an important medicinal plant, grows in hot and dry conditions, however, molecular mechanisms related to such adaptive properties are not known. Here, we elucidated that members of the sterol glycosyltransferases (SGT) gene family play important roles in the survival of W. somnifera under adverse conditions through maintaining the integrity of the membrane. SGTs are enzymes involved in sterol modifications and participate in metabolic flexibility during stress. Silencing of WsSGT members, for instance WsSGTL1, WsSGTL2 and WsSGTL4, was inimical for important physiological parameters, such as electron transport rate, photochemical quantum yield, acceptor side limitation, non‐photochemical quenching (NPQ), Fv/Fm and net photosynthetic rate, whereas stomatal conductance, transpiration rate and dark respiration rates (Rds) were increased. Decreased NPQ and increased Rds helped to generate significant amount of ROS in the Wsamisgt lines. After heat stress, H₂O₂, lipid peroxidation and nitric oxide production increased in the Wsamisgt lines due to high ROS generation. The expression of HSPs in Wsamisgt lines might be involved in regulation of physiological processes during stress. We have also observed increased proline accumulation which might be involved in restricting water loss in the Wsamisgt lines. Taken together, our observations revealed that SGTL enzyme activity is required to maintain the internal damages of the cell against high temperature by maintaining the sterol vs sterol glycosides ratio in the membranes of W. somnifera.     

Crag Is a GEF for Rab11 Required for Rhodopsin Trafficking and Maintenance of Adult Photoreceptor Cells

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.     

Identification of main effect and epistatic quantitative trait loci for morphological and yield-related traits in peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

An effort was made in the present study to identify the main effect and epistatic quantitative trait locus (QTL) for the morphological and yield-related traits in peanut. A recombinant inbred line (RIL) population derived from TAG 24 × GPBD 4 was phenotyped in seven environments at two locations. QTL analysis with available genetic map identified 62 main-effect QTLs (M-QTLs) for ten morphological and yield-related traits with the phenotypic variance explained (PVE) of 3.84–15.06%. Six major QTLs (PVE >?10%) were detected for PLHT, PPP, YPP, and SLNG. Stable M-QTLs appearing in at least two environments were detected for PLHT, LLN, YPP, YKGH, and HSW. Five M-QTLs governed two traits each, and 16 genomic regions showed co-localization of two to four M-QTLs. Intriguingly, a major QTL reported to be linked to rust resistance showed pleiotropic effect for yield-attributing traits like YPP (15.06%, PVE) and SLNG (13.40%, PVE). Of the 24 epistatic interactions identified across the traits, five interactions involved six M-QTLs. Three interactions were additive × additive and remaining two involved QTL × environment (QE) interactions. Only one major M-QTL governing PLHT showed epistatic interaction. Overall, this study identified the major M-QTLs for the important productivity traits and also described the lack of epistatic interactions for majority of them so that they can be conveniently employed in peanut breeding.     

Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: Design,synthesis, biological investigation and docking studies

In an attempt to develop potent anti-tubulin agents against most dreadful disease cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC₅₀ value of 1.06 µM which was further confirmed by using confocal microscopy. Possible binding interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by molecular modeling studies.