Environmental impact assessment via life cycle analysis on ultrafiltration membrane fabricated from polyethylene terephthalate waste to treat microalgal cultivation wastewater for reusability.

The current study had conducted the life cycle analysis (LCA) to assess the environmental impact of microalgal wastewater treatment via an integrated membrane bioreactor. The functional unit selected for this analysis was 1 kg of treated microalgal wastewater with contaminants eliminated by ultrafiltration membrane fabricated from recycled polyethylene terephthalate waste. Meanwhile, the applied system boundary in this study was distinguished based on two scenarios, namely, cradle-to-gate encompassed wastewater treatment only and cradle-to-cradle which included the reutilization of treated wastewater to cultivate microalgae again. The environmental impacts and hotspots associated with the different stages of the wastewater treatment process had clearly elucidated that membrane treatment had ensued the highest impact, followed by microalgal harvesting, and finally cultivation. Among the environmental impact categories, water-related impact was found to be prominent in the following series: freshwater ecotoxicity, freshwater eutrophication and marine ecotoxicity. Notably, the key performance indicator of all environmental impact, i.e., the global warming potential was found to be very much lower at 2.94 × 10-4 kg CO2 eq as opposed to other literatures reported on the LCA of wastewater treatments using membranes. Overall, this study had proffered insights into the environmental impact of microalgal wastewater treatment and its stimulus for sustainable wastewater management. The findings of this study can be instrumental in making informed decision for optimizing microalgal wastewater treatment and reutilization assisted by membrane technology with an ultimate goal of enhancing sustainability.

Factors affecting therapeutic protein purity and yield during chromatographic purification.

Therapeutic proteins are recombinant proteins generated through recombinant DNA technology and have attracted a great deal of interest in numerous applications, including pharmaceutical, cosmetic, human and animal health, agriculture, food, and bioremediation. Producing therapeutic proteins on a large scale, mainly in the pharmaceutical industry, necessitates a cost-effective, straightforward, and adequate manufacturing process. In industry, a protein separation technique based mainly on protein characteristics and modes of chromatography will be applied to optimize the purification process. Typically, the downstream process of biopharmaceutical operations may involve multiple chromatography phases that require the use of large columns pre-packed with resins that must be inspected before use. Approximately 20% of the proteins are assumed to be lost at each purification stage during the production of biotherapeutic products. Hence, to produce a high quality product, particularly in the pharmaceutical industry, the correct approach and understanding of the factors influencing purity and yield during purification are necessary.

Mechanistic behaviour of Chlorella vulgaris biofilm formation onto waste organic solid support used to treat palm kernel expeller in the recent Anthropocene.

The capacity to maximize the proliferation of microalgal cells by means of topologically textured organic solid surfaces under various pH gave rise to the fundamental biophysical analysis of cell-surface attachment in this study. The substrate used in analysis was palm kernel expeller (PKE) in which the microalgal cells had adhered onto its surface. The findings elucidated the relevance of surface properties in terms of surface wettability and surface energy in relation to the attached microalgal growth with pH as the limiting factor. The increase in hydrophobicity of PKE-microalgae attachment was able to facilitate the formation of biofilm better. The pH 5 and pH 11 were found to be the conditions with highest and lowest microalgal growths, respectively, which were in tandem with the highest contact angle value at pH 5 and conversely for pH 11. The work of attachment (Wcs) had supported the derived model with positive values being attained for all the pH conditions, corroborating the thermodynamic feasibility. Finally, this study had unveiled the mechanism of microalgal attachment onto the surface of PKE using the aid of extracellular polymeric surfaces (EPS) from microalgae. Also, the hydrophobic nature of PKE enabled excellent attachment alongside with nutrients for microalgae to grow and from layer-by-layer (LbL) assembly. This assembly was then isolated using organosolv method by means of biphasic solvents, namely, methanol and chloroform, to induce detachment.

Fundamental alteration of cellular biochemicals from attached microalgae onto palm kernel expeller waste upon optimizing the growth environment in forming adhesion complex.

Changing the growth environment for microalgae can overall lead to the fundamental alteration in cellular biochemicals whilst attaching onto palm kernel expeller (PKE) waste to form adhesion complex in easing harvesting at stationary growth phase. This study had initially optimized the PKE dosage, light intensity and photoperiod in maximizing the attached microalgal productivity being attained at 0.72 g/g day. Lipid content increased progressively from pH 3 to pH 11, with the highest value observed at pH 11. Meanwhile, in terms of protein and carbohydrate contents, the highest values were obtained by cultivation medium of pH 5 with 9.92 g and 17.72 g, respectively followed by pH 7 with 9.16 g and 16.36 g, respectively. Moreover, the findings also suggested that the low pH mediums utilized polar interactions in the formation of complexes between PKE and microalgae, whereas at higher pH levels, the non-polar interactions became more significant. The work of attachment was thermodynamically favourable towards the attachment formation with values greater than zero which was also aligned with the microscopic surface topography, i.e., revealing a clustering pattern of microalgae colonizing the PKE surface. These findings contribute to comprehensive understanding of optimizing growth condition and harvesting strategy of attached microalgae in attaining the cellular biochemical components, facilitating the development of efficient and sustainable bioresource utilization.

A phase 1b study of OXIRI in pancreatic adenocarcinoma patients and its immunomodulatory effects.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and debilitating disease with limited therapeutic options. The aim of this clinical study was to evaluate the safety, efficacy and pharmacokinetics of a novel regimen comprised of metronomic oxaliplatin (O), chronomodulated capecitabine (X) and UGT1A1 genotype-guided dosing of irinotecan (IRI) [OXIRI] as well as its immunomodulatory effects. Thirty-six patients were enrolled into either dose-escalation or expansion cohorts. In the dose escalation phase, capecitabine doses (2000, 2650, 3500 and 4500 mg/day) were administered at midnight on days 1 to 14 while oxaliplatin and irinotecan were intravenously infused at fixed doses of 50 and 75 mg/m2 respectively on days 1, 8 in a 21-day cycle. The maximum tolerated dose of capecitabine was 2650 mg/day and the most common grade 3 adverse events were neutropenia (30.6%) and diarrhea (13.9%). No grade 4 toxicity was observed. UGT1A1-genotype directed dosing resulted in similar exposure levels of irinotecan, SN-38 and SN-38G in all patients. Objective response rate was 22.2%. Median overall survival and progression-free survival were 8.1 and 5.2 months, respectively. Exploratory immunoprofiling by flow cytometry and quantitative spatial localization analysis of infiltrated immune cells performed on biopsy and plasma samples revealed significant declines in CCL22, CCL2 and TNFα levels at end of first cycle and an active host immune response. Our study showed that OXIRI was well-tolerated and exhibited good efficacy, with immunomodulatory effects. It may be considered as an alternative to FOLFIRINOX in patients intolerant to the latter.

Adaptation to an Amoeba Host Leads to Pseudomonas aeruginosa Isolates with Attenuated Virulence.

The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the environment, and in humans, it is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when coincubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors, and this correlated with a reduction in expression of virulence traits. Virulence for the nematode Caenorhabditis elegans was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and nonadapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoebae as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoebae resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water, where bacteria are constantly under threat of being consumed by bacterial predators, e.g., protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examined the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa. We show that long-term coincubation with protozoa gave rise to mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to those observed in isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures among host cell types as well as similar adaptation strategies.

Seroprevalence of Pteropine orthoreovirus in humans remain similar after nearly two decades (2001-2002 vs. 2017) in Tioman Island, Malaysia.

Pteropine orthoreovirus (PRV) is an emerging zoonotic respiratory virus that can be transmitted from bats to humans. In Malaysia, aside from PRV2P (Pulau virus) being isolated from Pteropus hypomelanus sampled in Tioman Island, PRV3M (Melaka virus), PRV4K (Kampar virus), and PRV7S (Sikamat virus) were all isolated from samples of patients who reported having a disease spectrum from acute respiratory distress to influenza-like illness and sometimes even with enteric symptoms such as diarrhea and abdominal pain. Screening of sera collected from human volunteers on Tioman Island in 2001-2002 demonstrated that 12.8% (14/109) were positive for PRV2P and PRV3M. Taking all these together, we aim to investigate the serological prevalence of PRV (including PRV4K and PRV7S) among Tioman Island inhabitants again with the assumption that the seroprevalence rate will remain nearly similar to the above reported if human exposure to bats is still happening in the island. Using sera collected from human volunteers on the same island in 2017, we demonstrated seroprevalence of 17.8% (28/157) against PRV2P and PRV3M, respectively. Seropositivity of 11.4% among Tioman Island inhabitants against PRV4K and PRV7S, respectively, was described in this study. In addition, the seroprevalence of 89.5% (17/19), 73.6% (14/19), 63.0% (12/19), and 73.6% (14/19) against PRV2P, PRV3M, PRV4K, and PRV7S, respectively, were observed among pteropid bats in the island. We revealed that the seroprevalence of PRV among island inhabitants remains nearly similar after nearly two decades, suggesting that potential spill-over events in bat-human interface areas in the Tioman Island. We are unclear whether such spillover was directly from bats to humans, as suspected for the PRV3M human cases, or from an intermediate host(s) yet to be identified. There is a high possibility of the viruses circulating among the bats as demonstrated by high seroprevalence against PRV in the bats.

Affective Recommender System for Pet Social Network.

In this new era, it is no longer impossible to create a smart home environment around the household. Moreover, users are not limited to humans but also include pets such as dogs. Dogs need long-term close companionship with their owners; however, owners may occasionally need to be away from home for extended periods of time and can only monitor their dogs' behaviors through home security cameras. Some dogs are sensitive and may develop separation anxiety, which can lead to disruptive behavior. Therefore, a novel smart home solution with an affective recommendation module is proposed by developing: (1) an application to predict the behavior of dogs and, (2) a communication platform using smartphones to connect with dog friends from different households. To predict the dogs' behaviors, the dog emotion recognition and dog barking recognition methods are performed. The ResNet model and the sequential model are implemented to recognize dog emotions and dog barks. The weighted average is proposed to combine the prediction value of dog emotion and dog bark to improve the prediction output. Subsequently, the prediction output is forwarded to a recommendation module to respond to the dogs' conditions. On the other hand, the Real-Time Messaging Protocol (RTMP) server is implemented as a platform to contact a dog's friends on a list to interact with each other. Various tests were carried out and the proposed weighted average led to an improvement in the prediction accuracy. Additionally, the proposed communication platform using basic smartphones has successfully established the connection between dog friends.

Residual palm kernel expeller as the support material and alimentation provider in enhancing attached microalgal growth for quality biodiesel production.

Albeit the biodiesel production from suspended microalgal system has gained immense interests in recent years, the domineering limitation of being economically infeasible has hindered this technology from partaking into a large-scale operation. To curtail this issue, attached growth system had been introduced by various studies; however, those were still unable to alleviate the socio-economic challenges faced in commercializing the microalgal biomass production. Thus, this study had developed a novel approach in cultivating-cum-harvesting attached Chlorella vulgaris sp. microalgae, whilst using solid organic waste of palm kernel expeller (PKE) as the supporting and alimentation material for microalgal biofilm formation. The effects of three variables, namely, PKE dosage, light intensity, and photoperiod, were initially modelled and later optimized using Response Surface Methodology tool. The derived statistical models could predict the growth performances of attached microalgal biomass and lipid productivity. The optimum growing condition was attained at PKE dosage of 5.67 g/L, light intensity of 197 µmol/m2 s and photoperiod of 8 light and 16 dark hours/cycle, achieving the microalgal density and lipid content of 9.87 ± 0.05 g/g and 3.39 ± 0.28 g/g, respectively, with lipid productivity of 29.6 mg/L day. This optimum condition had led to the intensification of biodiesel quality with a high percentage of monounsaturated fatty acid, i.e., oleic acid (C18:1), encompassing 81.86% of total fatty acid methyl ester components. Given that the positive acquisition of PKE as an excellent supporting material in enhancing the microalgal density and lipid productivity that had resulted in the commercially viable biodiesel quality, this study served as a novel revolution in augmenting the microalgae and solid waste utilities for sustainable energy generation.

An intronic FTO variant rs16952570 confers protection against thiopurine-induced myelotoxicities in multiethnic Asian IBD patients.

Thiopurines are used in the treatment of inflammatory bowel disease (IBD) but remain clinically challenging to manage due to wide interpatient variability in clinical outcomes and adverse events. Apart from genetic variants in thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15) genes, polymorphisms in FTO alpha-ketoglutarate dependent dioxygenase (FTO) were found predictive of thiopurine-induced leukopenia, albeit with conflicting results. To clarify the role of FTO variants in a multiethnic Asian IBD cohort, we recruited 149 patients on thiopurine-based therapy and genotyped two FTO variants p.Ala134Thr (rs79206939) and rs16952570 T > C using Sanger sequencing. FTO p.Ala134Thr (rs79206939) was non-polymorphic and absent whereas intronic rs16952570 T > C was equally prevalent in Chinese (22%) and Indians (18%) and higher in Malays (28%). Higher nadir white blood cell (WBC) and absolute neutrophil count (ANC) levels were observed in patients harboring FTO rs16952570 CC genotypes compared with TT carriers at 4, 8, and 12 weeks after start of thiopurine therapy (P < 0.05). A similar trend was observed in patients carrying the previously well-characterized NUDT15 rs116855232 wild-type CC genotypes. Further in silico analysis suggests that FTO variants linked to rs16952570, particularly rs74018601, may play a regulatory role in altering the FTO expression. The findings from this study indicate a novel protective association with the FTO variant rs16952570 CC genotype and hematological parameters.

Adaptogenic flower buds exert cancer preventive effects by enhancing the SCFA-producers, strengthening the epithelial tight junction complex and immune responses.

Microbiome therapy has attracted a keen interest from both research and business sectors. Our lab has been applying this "second genome" platform to assess the functionality of herbal medicines with fulfilling results. In this study, we applied this platform to assess the potential cancer-preventive effects of three selected adaptogenic plants. The flower buds from these plants were used to constitute Preparations SL and FSP according to the receipts of two commonly consumed Chinese medicinal decoctions for gastrointestinal discomfort. Preparation SL contains Sophorae japonica and Lonicerae Japonicae, and Preparation FSP contains Sophorae japonica and Gardenia Jasminoides. SL and FSP extracts significantly (p < 0.001) lowered the polyp burden, as well as the expressions of oncogenic signaling molecules, such as MAPK/ERK, PI3K/AKT, and STAT3 in ApcMin/+ mice. The inflamed gut was alleviated by shifting M1 to M2 macrophage phenotypes and the associated immune cytokines. The other remarkable change was on the extracellular tight junction protein complex, where the occludin, ZO-1, ICAM-1, E-cadherin were significantly (p < 0.05) upregulated while the N-cadherin and ß-catenin were downregulated in the treated mice. The above physiological changes in the gut epithelial barrier were companied with the changes in gut microbiome. The 16S Sequencing data revealed a marked decrease in the potential pathogens (especially Helicobacter species and hydrogen sulfide producing-bacteria) and the increase in beneficial bacteria (especially for species from the genera of Akkermansia, Barnesiella, Coprococcus, Lachnoclostridium, and Ruminococcus). The majority of which were the short-chain fatty acids (SCFAs) producers. Meanwhile SCFAs-sensing G protein-coupled receptors (GPCRs), including GPR41, GPR43, and GPR109a were also significantly upregulated. In a recent report, we proved that the bacteria-derived SCFAs plays an essential role to the anti-cancer effects of the mushroom polysaccharides and saponins in ApcMin/+ mice. In this study, we further demonstrated that butyrate treatment could enhance the extracellular tight junction protein complex as effective as the treatments with SL and FSP to the ApcMin/+ mice. Our findings provide strong evidence of the vital role of the SCFA-producers and their metabolites to the cancer-preventive properties of the SL and FSP preparations.

Comparing point shear wave elastography (ElastPQ) and transient elastography for diagnosis of fibrosis stage in non-alcoholic fatty liver disease.

BACKGROUND AND AIM: Transient elastography (TE) and point shear wave elastography (pSWE) are noninvasive methods to diagnose fibrosis stage in patients with chronic liver disease. The aim of this study is to compare the accuracy of the two methods to diagnose fibrosis stage in non-alcoholic fatty liver disease (NAFLD) and to study the intra-observer and inter-observer variability when the examinations were performed by healthcare personnel of different backgrounds. METHODS: Consecutive NAFLD patients who underwent liver biopsy were enrolled in this study and had two sets each of pSWE and TE examinations by a nurse and a doctor on the same day of liver biopsy procedure. The medians of the four sets of pSWE and TE were used for evaluation of diagnostic accuracy using area under receiver operating characteristic curve (AUROC). Intra-observer and inter-observer variability was analyzed using intraclass correlation coefficients. RESULTS: Data for 100 NAFLD patients (mean age 57.1 ± 10.2 years; male 46.0%) were analyzed. The AUROC of TE for diagnosis of fibrosis stage ≥ F1, ≥ F2, ≥ F3, and F4 was 0.89, 0.83, 0.83, and 0.89, respectively. The corresponding AUROC of pSWE was 0.80, 0.72, 0.69, and 0.79, respectively. TE was significantly better than pSWE for the diagnosis of fibrosis stages ≥ F2 and ≥ F3. The intra-observer and inter-observer variability of TE and pSWE measurements by the nurse and doctor was excellent with intraclass correlation coefficient > 0.96. CONCLUSION: Transient elastography was significantly better than pSWE for the diagnosis of fibrosis stage ≥ F2 and ≥ F3. Both TE and pSWE had excellent intra-observer and inter-observer variability when performed by healthcare personnel of different backgrounds.

Limited efficacy of diphenylcyclopropenone in the treatment of alopecia areata: Experience from a Tertiary Healthcare Institution in Singapore.

Alopecia areata (AA) is a common cause of nonscarring hair loss. Diphenylcyclopropenone (DPCP) is a form of contact immunotherapy used in the treatment of AA. We retrospectively reviewed all patients who were diagnosed with AA over a 4-year period (1st January 2012 to 31st December 2015) and who have received DPCP. Forty patients were studied in total. The mean duration of disease prior to the study was 195 days. Patients received a mean number of 14.91 sessions (range: 1-65). The mean number of sessions required before clinical response was seen was 2.33 sessions, corresponding to 0.001% DPCP. Based on the modified Global Assessment Grading System, 33.5% (n = 11) of the patients experienced less than 25% improvement, 48.5% (n = 16) experienced 25%-74% improvement and 18.3% (n = 6) experienced more than 75% improvement. One patient had severe sensitisation reaction amounting to near erythroderma which resolved completely upon cessation of DPCP therapy. No other adverse reactions were noted in the cohort. DPCP remains a valuable tool in a dermatologist's armamentarium in treating alopecia areata as it is safe, well-tolerated, and shows limited efficacy.

Transcriptogenomics identification and characterization of RNA editing sites in human primary monocytes using high-depth next generation sequencing data.

High-depth next generation sequencing data provide valuable insights into the number and distribution of RNA editing events. Here, we report the RNA editing events at cellular level of human primary monocyte using high-depth whole genomic and transcriptomic sequencing data. We identified over a ten thousand putative RNA editing sites and 69% of the sites were A-to-I editing sites. The sites enriched in repetitive sequences and intronic regions. High-depth sequencing datasets revealed that 90% of the canonical sites were edited at lower frequencies (<0.7). Single and multiple human monocytes and brain tissues samples were analyzed through genome sequence independent approach. The later approach was observed to identify more editing sites. Monocytes was observed to contain more C-to-U editing sites compared to brain tissues. Our results establish comparable pipeline that can address current limitations as well as demonstrate the potential for highly sensitive detection of RNA editing events in single cell type.

Mushroom polysaccharides and jiaogulan saponins exert cancer preventive effects by shaping the gut microbiota and microenvironment in ApcMin/+ mice.

The incidence of colorectal cancer (CRC) is alarming among younger peoples. While no effective chemopreventive drug available in the market, researchers have been searching for alternative strategies against CRC that are in demand. Therefore, we tested the cancer-preventive properties of Ganoderma lucidum (Lingzhi) polysaccharides (GLP), along with the saponins extracted from Gynostemma pentaphyllum (GpS), an herbal tea with prebiotic-like effects. Here, we report that saponins from Gynostemma pentaphyllum (GpS) and polysaccharides from Ganoderma lucidum (GLP together with GpS) profoundly improved the inflamed gut barrier of ApcMin/+ mice by reducing polyps, shifting colonic M1 to M2 macrophages, positively reverting E-cadherin/N-cadherin ratio, and downregulating oncogenic signaling molecules. The treatments also markedly promoted short-chain fatty acids (SCFAs)-producing bacteria and abridged sulfate-reducing bacteria in a time-dependent manner. G-protein coupled-receptors were significantly stimulated in the treated mice, accompanied by the modulated expressions of histone deacetylases, anti-cancer gut hormone PYY, and PPAPγ. These findings suggest that some of the herbal medicinal foods could modulate the relationship between the host and the gut microbiota (GM) to exert their beneficial properties to the host. Our study also implicates that these dietary mushroom polysaccharides and the Gp saponins have the potential to be developed as new preventive medicines against CRC.

Impact of various microalgal-bacterial populations on municipal wastewater bioremediation and its energy feasibility for lipid-based biofuel production.

The microalgal-bacterial co-cultivation was adopted as an alternative in making microbial-based biofuel production to be more feasible in considering the economic and environmental prospects. Accordingly, the microalgal-bacterial symbiotic relationship was exploited to enhance the microbial biomass yield, while bioremediating the nitrogen-rich municipal wastewater. An optimized inoculation ratio of microalgae and activated sludge (AS:MA) was predetermined and further optimization was performed in terms of different increment ratios to enhance the bioremediation process. The nitrogen removal was found accelerating with the increase of the increment ratios of inoculated AS:MA, though all the increment ratios had recorded a near complete total nitrogen removal (94-95%). In light of treatment efficiency and lipid production, the increment ratio of 0.5 was hailed as the best microbial population size in accounting the total nitrogen removal efficiency of 94.45%, while not compromising the lipid production of 0.241 g/L. Moreover, the cultures in municipal wastewater had attained higher biomass and lipid productions of 1.42 g/L and 0.242 g/L, respectively, as compared with the synthetic wastewater which were only 1.12 g/L (biomass yield) and 0.175 g/L (lipid yield). This was possibly due to the presence of trace elements which had contributed to the increase of biomass yield; thus, higher lipid attainability from the microalgal-bacterial culture. This synergistic microalgal-bacterial approach had been proven to be effective in treating wastewater, while also producing useful biomass for eventual lipid production with comparable net energy ratio (NER) value of 0.27, obtained from the life-cycle analysis (LCA) studies. Thereby, contributing towards long-term sustainability and possible commercialization of microbial-based biofuel production.

Transcriptome landscape of human primary monocytes at different sequencing depth.

Differential gene and transcript expression pattern of human primary monocytes from healthy young subjects were profiled under different sequencing depths (50M, 100M, and 200M reads). The raw data consisted of 1.3 billion reads generated from RNA sequencing (RNA-Seq) experiments. A total of 17,657 genes and 75,392 transcripts were obtained at sequencing depth of 200M. Total splice junction reads showed an even more significant increase. Comparative analysis of the expression patterns of immune-related genes revealed a total of 217 differentially expressed (DE) protein-coding genes and 50 DE novel transcripts, in which 40 DE protein-coding genes were related to the immune system. At higher sequencing depth, more genes, known and novel transcripts were identified and larger proportion of reads were allowed to map across splice junctions. The results also showed that increase in sequencing depth has no effect on the sequence alignment.

Latitudinal Clines in Temperature and Salinity Tolerance in Tidepool Copepods.

Local adaptation has been understudied in marine systems, but might be expected to be pronounced in the tidepool copepod Tigriopus californicus, which has a broad geographic range and extremely restricted dispersal. Tolerance to temperature and salinity was assessed in 14 populations over a 20° latitudinal range. Adaptive differentiation to temperature and salinity was found at scales as low as 5.6 km. Latitudinal clines were significant, with northern populations being more tolerant of low salinity and less tolerant of high temperature and high salinity. Both temperature and salinity tolerance were more closely associated with long-term thermal maxima than with long-term precipitation data. Hyperthermal and hyposmotic tolerance were inversely correlated, a pattern that could potentially slow adaptation to future conditions. Together, these studies of intraspecific geographic patterns in resistance to multiple stressors are important in predicting how environmental change may effect range shifts and local extinctions.

Flicking technique for microencapsulation of cells in calcium alginate leading to the microtissue formation.

Microbeads have wide applications in biomedical engineering field that include drug delivery, encapsulation of biomolecules, tissue padding and tissue regeneration. In this paper, we report a simple, yet efficient, flicking technique to produce microcapsules of calcium alginate at a narrow distribution of size. The system consists of an infusion pump and a customised flicker that taps the syringe needle for dispersing microcapsules of sodium alginate that polymerised in the calcium chloride solution. The flow rate of the syringe pump and the velocity of the flicker were studied to achieve a well controlled and tunable size distribution of microbeads ranging from 200 to 400 µm. At a flow rate of 4 µl/min and flicking rate of 80 rpm, a narrow size distribution of microbeads were produced. Via this technique, HaCaT cells were encapsulated in calcium alginate microbeads that grown into microtissues with a size ranging from 100 to 300 µm after two weeks of culture. These microtissues could be potentially useful for pharmacological application.