Silver Nanoparticles from Oregano Leaves' Extracts as Antimicrobial Components for Non-Infected Hydrogel Contact Lenses.

The oregano leaves' extract (ORLE) was used for the formation of silver nanoparticles (AgNPs(ORLE)). ORLE and AgNPs(ORLE) (2 mg/mL) were dispersed in polymer hydrogels to give the pHEMA@ORLE_2 and pHEMA@AgNPs(ORLE)_2 using hydroxyethyl-methacrylate (HEMA). The materials were characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), thermogravimetric differential thermal analysis (TG-DTA), derivative thermogravimetry/differential scanning calorimetry (DTG/DSC), ultraviolet (UV-Vis), and attenuated total reflection mode (ATR-FTIR) spectroscopies in solid state and UV-Vis in solution. The crystallite size value, analyzed with XRPD, was determined at 20 nm. The antimicrobial activity of the materials was investigated against Gram-negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). The Gram-positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) are known to be involved in microbial keratitis by the means of inhibitory zone (IZ), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The IZs, which developed upon incubation of P. aeruginosa, E. coli, S. epidermidis, and S. aureus with paper discs soaked in 2 mg/mL of AgNPs(ORLE), were 11.7 ± 0.7, 13.5 ± 1.9, 12.7 ± 1.7, and 14.3 ± 1.7 mm. When the same dose of ORLE was administrated, the IZs were 10.2 ± 0.7, 9.2 ± 0.5, 9.0 ± 0.0, and 9.0 ± 0.0 mm. The percent of bacterial viability when they were incubated over the polymeric hydrogel discs of pHEMA@AgNPs(ORLE)_2 was interestingly low (66.5, 88.3, 77.7, and 59.6%, respectively, against of P. aeruginosa, E. coli, S. epidermidis, and S. aureus) and those of pHEMA@ORLE_2 were 89.3, 88.1, 92.8, and 84.6%, respectively. Consequently, pHEMA@AgNPs(ORLE)_2 could be an efficient candidate toward the development of non-infectious contact lenses.

Biocontrol efficacy of Trichoderma asperellum-enriched coconut fibre against Fusarium wilts of cherry tomato.

AIMS: Agro-based wastes were evaluated as a medium for mass micropropagule production and optimal efficacy of Trichoderma asperellum B1092 in controlling Fusarium oxysporum f. sp. lycopersici and promoting tomato growth. This study focused on biological control because pathogen persistence in the soil makes the disease difficult to control. METHODS AND RESULTS: Rice bran, biochar, empty fruit bunches, coconut fibres, compost, top soil and mixed soil were evaluated as media for mass multiplication of T. asperellum, which is effective in controlling plant pathogens. Yielding the most colony forming units (CFU) among the media, coconut fibre was deemed most suitable for promoting sporulation. After 120 days on the medium, T. asperellum B1902 produced 9·053 × 105  CFU per gram coconut fibre; oil palm empty fruit bunches was second highest (7·406 × 105  CFU per gram). In field tests of T. asperellum B1092 against F. oxysporum f. sp lycopersici (causing Fusarium wilt of cherry tomato), B1092 significantly promoted plant growth compared to the control. The efficacy of this formulation resulted in increased growth of roots and shoots tomato plants and total lycopene, sugar, K, N, Ca, P and Mg content after 120 days. CONCLUSIONS: Trichoderma asperellum B1092 showed great field potential for improving productivity and quality of tomatoes and in controlling Fusarium wilt of cherry tomato. SIGNIFICANCE AND IMPACT OF THE STUDY: This innovative approach using a cheap agro-waste to control the persistent soil-borne Fusarium pathogen of cherry tomato should increase soil survival rate of Trichoderma and has potential for upscaling in the field for other crops.

Bioavailability and mobility of arsenic, cadmium, and manganese in gold mine tailings amended with rice husk ash and Fe-coated rice husk ash.

This study was conducted to determine the effects of rice husk ash (RHA) and Fe-coated rice husk ash (Fe-RHA) on the bioavailability and mobility of As, Cd, and Mn in mine tailings. The amendments were added to the tailings at 0, 5, 10, or 20% (w/w) and the mixtures were incubated for 0, 7, 15, 30, 45, and 60 days. The CaCl2 extractable As, Cd, and Mn in the amended tailings were determined at each interval of incubation period. In addition, the tailings mixture was leached with simulated rain water (SRW) every week from 0 day (D 0) until day 60 (D 60). The results showed that both RHA and Fe-RHA application significantly decreased the CaCl2-extractable Cd and Mn but increased that of As in the tailings throughout the incubation period. Consequently, addition of both RHA and Fe-RHA leached out higher amount of As from the tailings but decreased Cd and Mn concentration compared to the controls. The amount of As leached from the Fe-RHA-amended tailings was less than that from RHA-amended tailings. Application of both RHA and Fe-RHA could be an effective way in decreasing the availability of cationic heavy metals (Cd and Mn) in the tailings but these amendments could result in increasing the availability of anionic metalloid (As). Therefore, selection of organic amendments to remediate metal-contaminated tailings must be done with great care because the outcomes might be different among the elements.

Changes in gene expression in the rumen and colon epithelia during the dry period through lactation of dairy cows and effects of live yeast supplementation.

The objectives of this study were (1) to use endoscopy to collect biopsies from the rumen and colon epithelia to describe changes in gene expression in these 2 tissues as cows move from a dry to a lactation ration and (2) to evaluate the potential influence that supplementation of live yeast could exert on these 2 epithelia. Twenty-one Holstein cows were split into 2 treatments and received either 300 g/d of corn containing 1 × 1010 cfu/d of live yeast (LY; n = 10) or 300 g/d of corn with no supplementation (control; n = 11) starting 21 ± 2.6 d (average ± SD) before until 21 d after calving. At 14 ± 2.6 d before the expected calving date, and exactly at 7 and 21 d after calving, rumen and colon biopsies were obtained from each cow using an endoscope. Total RNA was extracted from rumen and colon tissues, and the expression of IL10, TNFA, TLR4, IL1B, PCNA, MKI67, SGLT1, BAX, CASP3, OCLN, CLDN4, HSPA1A, HSPB1, DEFB1, and MCT1 (the latter only in rumen samples) was quantified by quantitative PCR. Overall, fluctuations in expression of the selected genes in the colon between the 2 stages of production and the 2 treatments were smaller than those found in the rumen. In the rumen epithelium, expression of TLR4 and DEFB1 was greatest before calving, with LY cows having a greater expression of TLR4 than control cows. Similarly, expression of IL10 was greatest in LY cows before calving. Expression of TNFA in the rumen epithelium of control cows was lowest at 21 DIM but in LY cows was kept steady among production stages. The expression of PCNA and MKI67 in the rumen epithelium was greatest at 7 DIM, indicating a high proliferation rate of this epithelium after calving. In the colon mucosa, expression of TLR4 and DEFB1 was greater than in the rumen, and DEFB1 expression was greater in LY cows than in control cows. The use of an endoscope allowed us to study the dynamics of rumen epithelium adaptation to increased supply of concentrate after calving, consisting of increased epithelia remodeling, reduction of the TLR4, and increased IL10 expression. Furthermore, the rumen epithelium of dry cows responded rapidly to live yeast, with changes in the expression of genes involved in the immune response becoming evident after 7 d of exposure to yeast. The expression of genes related to the immune response (mainly TLR4 and DEFB1) in the colon mucosa was greater than in the rumen, and the expression of DEFB1 was further stimulated by live yeast. It is concluded that the use of an endoscope allows the study of gene expression patterns in the rumen and hindgut epithelia. We report marked changes in the rumen wall and more modest changes in the colon when transitioning from a dry to a lactation ration. Furthermore, supplementation of live yeast fostered and increased expression of genes regulating inflammation and epithelial barrier in the rumen, and in the colon it increased the expression of DFEB1 coding for an antimicrobial peptide.

Low-dissolved-oxygen nitrification in tropical sewage: an investigation on potential, performance and functional microbial community.

Intensive aeration for nitrification is a major energy consumer in sewage treatment plants (STPs). Low-dissolved-oxygen (low-DO) nitrification has the potential to lower the aeration demand. However, the applicability of low-DO nitrification in the tropical climate is not well-understood. In this study, the potential of low-DO nitrification in tropical setting was first examined using batch kinetic experiments. Subsequently, the performance of low-DO nitrification was investigated in a laboratory-scale sequential batch reactor (SBR) for 42 days using real tropical sewage. The batch kinetic experiments showed that the seed sludge has a relatively high oxygen affinity. Thus, the rate of nitrification was not significantly reduced at low DO concentrations (0.5 mg/L). During the operation of the low-DO nitrification SBR, 90% of NH4-N was removed. The active low-DO nitrification was mainly attributed to the limited biodegradable organics in the sewage. Fluorescence in-situ hybridisation and 16S rRNA amplicon sequencing revealed the nitrifiers were related to Nitrospira genus and Nitrosomonadaceae family. Phylogenetic analysis suggests 47% of the operational taxonomic units in Nitrospira genus are closely related to a comammox bacteria. This study has demonstrated active low-DO nitrification in tropical setting, which is a more sustainable process that could significantly reduce the energy footprint of STPs.

Status, source identification, and health risks of potentially toxic element concentrations in road dust in a medium-sized city in a developing country.

This study aims to determine the status of potentially toxic element concentrations of road dust in a medium-sized city (Rawang, Malaysia). This study adopts source identification via enrichment factor, Pearson correlation analysis, and Fourier spectral analysis to identify sources of potentially toxic element concentrations in road dust in Rawang City, Malaysia. Health risk assessment was conducted to determine potential health risks (carcinogenic and non-carcinogenic risks) among adults and children via multiple pathways (i.e., ingestion, dermal contact, and inhalation). Mean of potentially toxic element concentrations were found in the order of Pb > Zn > Cr(IV) > Cu > Ni > Cd > As > Co. Source identification revealed that Cu, Cd, Pb, Zn, Ni, and Cr(IV) are associated with anthropogenic sources in industrial and highly populated areas in northern and southern Rawang, cement factories in southern Rawang, as well as the rapid development and population growth in northwestern Rawang, which have resulted in high traffic congestion. Cobalt, Fe, and As are related to geological background and lithologies in Rawang. Pathway orders for both carcinogenic and non-carcinogenic risks are ingestion, dermal contact, and inhalation, involving adults and children. Non-carcinogenic health risks in adults were attributed to Cr(IV), Pb, and Cd, whereas Cu, Cd, Cr(IV), Pb, and Zn were found to have non-carcinogenic health risks for children. Cd, Cr(IV), Pb, and As may induce carcinogenic risks in adults and children, and the total lifetime cancer risk values exceeded incremental lifetime.

Associations between subclinical hypocalcemia and postparturient diseases in dairy cows.

Dairy cows suffer blood Ca losses as lactation begins and might be affected by hypocalcemia in its clinical (total serum Ca concentration <1.50 mM) or subclinical form (total serum Ca concentration ≤2.14 mM). Several studies have suggested that hypocalcemia is associated with different health problems of the cow but results from different studies are not consistent. The objective of this study was to assess potential associations between subclinical hypocalcemia (SCHC) and displaced abomasum, intramammary infections, metritis, retained placenta, and ketosis. Also, the associations between SCHC and milk yield and reproductive function were evaluated. After discarding cows (32) with clinical hypocalcemia, a total of 764 cows from 6 different commercial farms were enrolled in this study. Blood samples were collected at 24 to 48 h postcalving and analyzed for total Ca concentration. Odds ratios of the different afflictions potentially associated with SCHC were calculated. Seventy-eight percent of the analyzed cows incurred SCHC. The occurrence of displaced abomasum, ketosis, retained placenta, and metritis was 3.7, 5.5, 3.4, and 4.3 times more likely, respectively, in cows that had SCHC than in cows with normocalcemia. Furthermore, the risk of incurring retained placenta or metritis increased in multiparous cows as serum Ca concentrations decreased compared with that in primiparous cows. Normocalcemic cows, independent of parity, were more likely to show their first estrus sooner after calving than SCHC cows, but no correlation was found between SCHC and other reproductive parameters. Different serum Ca concentration cutoffs were identified for several postpartum afflictions (≤1.93, ≤2.05, ≤2.05, and ≤2.10 mM for ketosis, retained placenta, metritis, and displaced abomasum, respectively). In conclusion, SCHC, defined as serum Ca ≤2.14 mM, is a frequent illness affecting the majority of the dairy cows with important repercussions on health. However, if SCHC were to be used to predict postpartum disease, different serum Ca cutoff points are likely to be needed because best predictive cutoff values varied among postpartum ketosis, displaced abomasum, retained placenta, and metritis.

Is calcitonin an active hormone in the onset and prevention of hypocalcemia in dairy cattle?

The objective of this study was to assess the potential importance of calcitonin (CALC) in the onset of subclinical hypocalcemia (experiment 1) and in the physiological mechanisms underlying the prevention of bovine hypocalcemia under metabolic acidosis (experiments 2 and 3). In experiment 1, 15 Holstein cows naturally incurring subclinical hypocalcemia during the first 5d postpartum were classified as low subclinical hypocalcemia (LSH) when blood Ca concentrations were between 7.5 and 8.5mg/dL, or as high subclinical hypocalcemia (HSH) when blood Ca concentrations were between 6.0 and 7.6 mg/dL. Blood samples were taken daily from d -5 to 5 relative to parturition to determine concentrations of parathyroid hormone (PTH), CALC, and 1,25(OH)2D3. In experiment 2, 24 Holstein bulls (497 ± 69 kg of body weight and 342 ± 10.5d of age) were assigned to 2 treatments (metabolic acidosis or control). Metabolic acidosis was induced by an oral administration of ammonium chloride (2.5 mEq/d) during 10 d, and animals were slaughtered thereafter. Blood samples were collected before slaughter to determine CALC, PTH, 1,25(OH)2D3, and samples of urine, kidney, parathyroid, and thyroid glands were obtained immediately after slaughter to determine expression of several genes in these tissues. Last, in experiment 3, we tested the activity of CALC under metabolic acidosis in vitro using breast cancer cell (T47D) cultures. Although PTH tended to be greater in HSH than in LSH, the levels of 1,25(OH)2D3 were lower in HSH cows (experiment 1). Blood CALC concentration was not affected by the severity of subclinical hypocalcemia, but it was influenced by days from calving (experiment 1). The expression of PTH receptor (PTHR) in the kidney was increased under metabolic acidosis (experiment 2). Furthermore, the activity of CALC was impaired under acidic blood pH (experiment 3). In conclusion, the CALC rise in HSH cows after calving impaired the recovery of blood Ca concentrations because the PTHR response was not sufficient to activate 1,25(OH)2D3 and compensate for the CALC effect. Metabolic acidosis prevents hypocalcemia because the expression of PTHR is upregulated in the kidney, resulting in an increased PTH activity and a subsequent increase in 1,25(OH)2D3 serum concentrations. Moreover, an impairment of CALC activity at low pH enhances the hypercalcemic role of PTH.

Quality of Kelantan drinking water and knowledge, attitude and practice among the population of Pasir Mas, Malaysia.

OBJECTIVES: Information about the quality of drinking water, together with analysis of knowledge, attitude and practice (KAP) analysis and health risk assessment (HRA) remain limited. The aims of this study were: (1) to ascertain the level of KAP regarding heavy metal contamination of drinking water in Pasir Mas; (2) to determine the concentration of heavy metals (Al, Cr, Cu, Fe, Ni, Pb, Zn and Cd) in drinking water in Pasir Mas; and (3) to estimate the health risks (non-carcinogenic and carcinogenic) caused by heavy metal exposure through drinking water using hazard quotient and lifetime cancer risk. METHODS: Information on KAP was collected using a standardized questionnaire. Heavy metal analysis of drinking water samples was performed using graphite furnace atomic absorption spectrophotometry. RESULTS: The population of Pasir Mas has good knowledge (80%), a less positive attitude (93%) and good practice (81%) towards heavy metal contamination of drinking water. The concentrations of heavy metals analysed in this study were found to be below the permissible limits for drinking water set by the Malaysian Ministry of Health and the World Health Organization. The HRA showed no potential non-carcinogenic and carcinogenic risks from the intake of heavy metal through drinking water. CONCLUSION: By investigating the quality of drinking water, KAP and HRA, the results of this study will provide authorities with the knowledge and resources to improve the management of drinking water quality in the future.

Short communication: The effects of cabergoline administration at dry-off of lactating cows on udder engorgement, milk leakages, and lying behavior.

Cabergoline is an ergot derivative with high affinity for the D2 dopamine receptors whose dopaminergic effects cause inhibition of prolactin (PRL) secretion; thus, it could be considered a molecule that acts as a potential dry-off facilitator. One hundred ninety-nine Holstein cows (102 primiparous; 97 multiparous) producing ≥ 18 kg/d at dry-off were split in 2 treatments to evaluate the effects of diminishing PRL secretion at dry-off (between 70 and 50 d from the expecting calving date) on udder engorgement, milk leakage, and cow well-being after dry-off. Treatments consisted of a single i.m. injection of 5 mL of a solution containing 5.6 mg of cabergoline (CAB; Velactis, Ceva Santé Animal, Libourne, France) or 5 mL of saline solution as a placebo (CTRL). Each animal was evaluated for presence or absence of milk leakages during the 4 d following dry-off and udder engorgement was determined using a digital algometer. Lying behavior was recorded during 10 d after dry-off. Twenty-five cows from each treatment were randomly chosen and blood sampled at 3 and 15 d after dry-off, and again at 5 and 3 d before the expected calving date to determine serum PRL concentrations. Cows on CAB had lower serum PRL concentrations than cows on CTRL at 3 and 15 d after dry-off. Average udder engorgement was lower for cows on CAB than for cows on CTRL following dry-off, and it decreased as days after dry-off increased. The overall incidence of milk leakage in cows on CAB (3.1 ± 0.88%) was 73.5% of that obtained in cows on CTRL (11.7 ± 1.64%); cows on CAB had 0.2 lower odds of incurring milk leakage than cows on CTRL. The day following dry-off, CAB cows lied about 1.5h/d more than cows on CTRL. We conclude that i.m. administration of 5.6 mg of cabergoline at dry-off effectively reduces PRL secretion, udder engorgement, and milk leakages, and improves lying time the day following dry-off.

Analysis of human invasive cytotrophoblasts using multicolor fluorescence in situ hybridization.

Multicolor fluorescence in situ hybridization, or FISH, is a widely used method to assess fixed tissues or isolated cells for numerical and structural chromosome aberrations. Unlike other screening procedures which provide average chromosome numbers for heterogeneous samples, FISH is a sensitive cell-by-cell method to analyze the distribution of abnormal cells in complex tissues. Here, we applied FISH to characterize chromosomal composition of a rare, but very important class of human cells that stabilize the fetal-maternal interface connecting the placenta to the uterine wall during early pregnancy, called invasive cytotrophoblasts (iCTBs). Combining differently-labeled, chromosome-specific DNA probes, we were able to unambiguously determine the number of up to six different autosomes and gonosomes in individual cell nuclei from iCTBs selected on the basis of their invasive behavior. In this manuscript, we describe a method for generation of iCTBs from placental villi, and provide the complete workflow of our FISH experiments including a detailed description of reagents and a trouble-shooting guide. We also include an in-depth discussion of the various types and sources of DNA probes which have evolved considerably in the last two decades. Thus, this communication represents both a complete guide as well as a valuable resource, intended to allow an average laboratory to reproduce the experiments and minimize the amount of specialized, and often costly, equipment.

Mammary serum amyloid A3 activates involution of the mammary gland in dairy cows.

The dry period is a nonlactating phase in which senescent mammary cells are regenerated, which is thought to optimize milk production in the subsequent lactation. In bovines, the dry period normally coexists with pregnancy and the lactogenic hormones delay mammary gland involution and impair the activation of immune system to fight the risk of intramammary infections. Conventionally, long dry periods of up to 60 d are required to allow sufficient mammary regeneration for full milk yield in the next lactation. The aim of this study was to evaluate the potential of mammary serum amyloid A3 (M-SAA3) as an activator of the involution of the mammary gland. One milligram of recombinant M-SAA3 and the corresponding negative controls (saline solution and lipopolysaccharide) were infused into the mammary gland via the teat canal, and mammary secretion samples were taken during the first 3 d after drying off to analyze metalloproteinase activity, somatic cell count, protein, and fat contents. Primary mammary gland epithelial cell cultures and bovine dendritic cells, obtained from necropsy tissue and blood, respectively, were incubated with and without M-SAA3 and cytokine expression was quantified. Last, the protective role of the M-SAA3 against infections was evaluated after a Staphylococcus aureus challenge. Matrix metalloproteinase 9 activity, a key protein that directly participates in the onset of the involution process, was greater in quarters treated with the M-SAA3. Protein content was increased in mammary secretions compared with control quarters. M-SAA3 increased cytokines directly related to innate immunity in both epithelial and dendritic cells and reduced the infection by Staphylococcus aureus.

Effects of forage provision to young calves on rumen fermentation and development of the gastrointestinal tract.

Fifteen Holstein male calves were randomly assigned to 1 of 3 dietary treatments according to age and body weight (BW) to determine the effects of feeding different forages sources on rumen fermentation and gastrointestinal tract (GIT) development. Treatments consisted of a starter (20% crude protein, 21% neutral detergent fiber) fed alone (CON) or supplemented with alfalfa (AH) or with oat hay (OH). All calves received 2L of milk replacer (MR) at 12.5% dry matter twice daily until 49 d of age. Calves received 2L of the same MR from 50 to 56 d of age and were weaned at 57 d of age. Individual starter, forage, and MR intakes were recorded daily and BW was recorded weekly. A rumen sample was taken weekly to determine rumen pH and volatile fatty acid concentrations. Three weeks after weaning, animals were harvested and each anatomical part of the GIT was separated and weighed with and without contents. Rumen pH was lower in CON than in OH and AH calves. Furthermore, acetate proportion in the rumen liquid tended to be greater in AH than in CON and OH treatments. Total GIT weight, expressed as a percentage of BW, tended to be greater in AH compared with the other 2 treatments. Rumen tissue tended to weigh more in CON than in OH animals. Animals with access to forage tended to have a greater expression of monocarboxylate transporter 1 than CON calves. In conclusion, calves supplemented with oat hay have a better rumen environment than calves offered no forage and do not have an increased gut fill.

Short communication: The biological value of transition milk: analyses of immunoglobulin G, IGF-I and lactoferrin in primiparous and multiparous dairy cows.

Colostrum (the first mammary gland secretion after calving) is known to contain high concentrations of nutrients as well as bioactive substances (including immunoglobulins, growth factors, and antimicrobial factors) to ensure neonatal survival. Due to its immunomodulatory, antibacterial, and antiviral activities, bovine colostrum has been used not only in calves but also in the prevention and treatment of human gastrointestinal and respiratory infections. Transition milk is the mammary secretion from the second milking to the sixth, which may contain these bioactive compounds to a lesser extent. The objective of the present study was to measure IGF-I, immunoglobulin G (IgG), and lactoferrin (LTF) concentrations in colostrum and transition milk of primiparous and multiparous cows to further assess its potential use in veterinary and nutraceutical applications. The results demonstrated that the concentrations of these three bioactive molecules decrease from the first milking to the tenth. Concentrations of IGF-I and LTF were greater in multiparous than in primiparous cows. Also, lactation number interacted with milking number in IGF-I, since primiparous cows had a smoother decline of IGF-I concentrations than multiparous ones. Overall, transition milk from the second milking showed a 46% decrease in the analysed colostrum bioactive molecules. Therefore, further studies are needed to apply this knowledge in neonate farm management practices or in developing pharmaceutical supplements from farm surpluses.

Bioinformatic Tools Identify Chromosome-Specific DNA Probes and Facilitate Risk Assessment by Detecting Aneusomies in Extra-embryonic Tissues.

Despite their non-diseased nature, healthy human tissues may show a surprisingly large fraction of aneusomic or aneuploid cells. We have shown previously that hybridization of three to six non-isotopically labeled, chromosome-specific DNA probes reveals different proportions of aneuploid cells in individual compartments of the human placenta and the uterine wall. Using fluorescence in situ hybridization, we found that human invasive cytotrophoblasts isolated from anchoring villi or the uterine wall had gained individual chromosomes. Chromosome losses in placental or uterine tissues, on the other hand, were detected infrequently. A more thorough numerical analysis of all possible aneusomies occurring in these tissues and the investigation of their spatial as well as temporal distribution would further our understanding of the underlying biology, but it is hampered by the high cost of and limited access to DNA probes. Furthermore, multiplexing assays are difficult to set up with commercially available probes due to limited choices of probe labels. Many laboratories therefore attempt to develop their own DNA probe sets, often duplicating cloning and screening efforts underway elsewhere. In this review, we discuss the conventional approaches to the preparation of chromosome-specific DNA probes followed by a description of our approach using state-of-the-art bioinformatics and molecular biology tools for probe identification and manufacture. Novel probes that target gonosomes as well as two autosomes are presented as examples of rapid and inexpensive preparation of highly specific DNA probes for applications in placenta research and perinatal diagnostics.

Chromosome-specific DNA repeats: rapid identification in silico and validation using fluorescence in situ hybridization.

Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as "database mining". Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

Optimizing the coagulation process in a drinking water treatment plant -- comparison between traditional and statistical experimental design jar tests.

In this study of coagulation operation, a comparison was made between the optimum jar test values for pH, coagulant and coagulant aid obtained from traditional methods (an adjusted one-factor-at-a-time (OFAT) method) and with central composite design (the standard design of response surface methodology (RSM)). Alum (coagulant) and polymer (coagulant aid) were used to treat a water source with very low pH and high aluminium concentration at Sri-Gading water treatment plant (WTP) Malaysia. The optimum conditions for these factors were chosen when the final turbidity, pH after coagulation and residual aluminium were within 0-5 NTU, 6.5-7.5 and 0-0.20 mg/l respectively. Traditional and RSM jar tests were conducted to find their respective optimum coagulation conditions. It was observed that the optimum dose for alum obtained through the traditional method was 12 mg/l, while the value for polymer was set constant at 0.020 mg/l. Through RSM optimization, the optimum dose for alum was 7 mg/l and for polymer was 0.004 mg/l. Optimum pH for the coagulation operation obtained through traditional methods and RSM was 7.6. The final turbidity, pH after coagulation and residual aluminium recorded were all within acceptable limits. The RSM method was demonstrated to be an appropriate approach for the optimization and was validated by a further test.

A Double-Pronged Sword: XJB-5-131 Is a Suppressor of Somatic Instability and Toxicity in Huntington's Disease.

Due to large increases in the elderly populations across the world, age-related diseases are expected to expand dramatically in the coming years. Among these, neurodegenerative diseases will be among the most devastating in terms of their emotional and economic impact on patients, their families, and associated subsidized health costs. There is no currently available cure or rescue for dying brain cells. Viable therapeutics for any of these disorders would be a breakthrough and provide relief for the large number of affected patients and their families. Neurodegeneration is accompanied by elevated oxidative damage and inflammation. While natural antioxidants have largely failed in clinical trials, preclinical phenotyping of the unnatural, mitochondrial targeted nitroxide, XJB-5-131, bodes well for further translational development in advanced animal models or in humans. Here we consider the usefulness of synthetic antioxidants for the treatment of Huntington's disease. The mitochondrial targeting properties of XJB-5-131 have great promise. It is both an electron scavenger and an antioxidant, reducing both somatic expansion and toxicity simultaneously through the same redox mechanism. By quenching reactive oxygen species, XJB-5-131 breaks the cycle between the rise in oxidative damage during disease progression and the somatic growth of the CAG repeat which depends on oxidation.

Flap Endonuclease 1 Endonucleolytically Processes RNA to Resolve R-Loops through DNA Base Excision Repair.

Flap endonuclease 1 (FEN1) is an essential enzyme that removes RNA primers and base lesions during DNA lagging strand maturation and long-patch base excision repair (BER). It plays a crucial role in maintaining genome stability and integrity. FEN1 is also implicated in RNA processing and biogenesis. A recent study from our group has shown that FEN1 is involved in trinucleotide repeat deletion by processing the RNA strand in R-loops through BER, further suggesting that the enzyme can modulate genome stability by facilitating the resolution of R-loops. However, it remains unknown how FEN1 can process RNA to resolve an R-loop. In this study, we examined the FEN1 cleavage activity on the RNA:DNA hybrid intermediates generated during DNA lagging strand processing and BER in R-loops. We found that both human and yeast FEN1 efficiently cleaved an RNA flap in the intermediates using its endonuclease activity. We further demonstrated that FEN1 was recruited to R-loops in normal human fibroblasts and senataxin-deficient (AOA2) fibroblasts, and its R-loop recruitment was significantly increased by oxidative DNA damage. We showed that FEN1 specifically employed its endonucleolytic cleavage activity to remove the RNA strand in an R-loop during BER. We found that FEN1 coordinated its DNA and RNA endonucleolytic cleavage activity with the 3'-5' exonuclease of APE1 to resolve the R-loop. Our results further suggest that FEN1 employed its unique tracking mechanism to endonucleolytically cleave the RNA strand in an R-loop by coordinating with other BER enzymes and cofactors during BER. Our study provides the first evidence that FEN1 endonucleolytic cleavage can result in the resolution of R-loops via the BER pathway, thereby maintaining genome integrity.

An infrared spectral biomarker accurately predicts neurodegenerative disease class in the absence of overt symptoms.

Although some neurodegenerative diseases can be identified by behavioral characteristics relatively late in disease progression, we currently lack methods to predict who has developed disease before the onset of symptoms, when onset will occur, or the outcome of therapeutics. New biomarkers are needed. Here we describe spectral phenotyping, a new kind of biomarker that makes disease predictions based on chemical rather than biological endpoints in cells. Spectral phenotyping uses Fourier Transform Infrared (FTIR) spectromicroscopy to produce an absorbance signature as a rapid physiological indicator of disease state. FTIR spectromicroscopy has over the past been used in differential diagnoses of manifest disease. Here, we report that the unique FTIR chemical signature accurately predicts disease class in mouse with high probability in the absence of brain pathology. In human cells, the FTIR biomarker accurately predicts neurodegenerative disease class using fibroblasts as surrogate cells.