Defined concatenated α6α1ß3γ2 GABAA receptor constructs reveal dual action of pyrazoloquinolinone allosteric modulators.

Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABAA receptors with different subunit composition, exerting modulatory effects by binding at α+/ß- interfaces of GABAA receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABAA receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands' R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6ß3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/ß3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/ß3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability.

Differential interference contrast microscopy for cells using hard x-ray holography.

We propose a differential interference contrast method for cells using hard x-ray Gabor holography and knife-edge filtering in the spatial frequency domain, without relying on beam shearing. A phase object is holographically recorded and reconstructed by computer. Interference between the wavefronts of zeroth order weighted by ejπ/2 in the positive frequency region produces a dark image. Similarly, interference between the wavefronts of the zeroth order weighted by ej3π/2 in the negative frequency region produces a bright image. By adding these two intensity distributions, good quality phase-contrast images of 8-µm-diameter polystyrene beads and human HeLa cells were obtained.

Genetic heterogeneity in Pakistani microcephaly families revisited.

Autosomal recessive primary microcephaly (MCPH) is a rare and heterogeneous genetic disorder characterized by reduced head circumference, low cognitive prowess and, in general, architecturally normal brains. As many as 14 different loci have already been mapped. We recruited 35 MCPH families in Pakistan and could identify the genetic cause of the disease in 31 of them. Using homozygosity mapping complemented with whole-exome, gene panel or Sanger sequencing, we identified 12 novel mutations in 3 known MCPH-associated genes - 9 in ASPM, 2 in MCPH1 and 1 in CDK5RAP2. The 2 MCPH1 mutations were homozygous microdeletions of 164,250 and 577,594 bp, respectively, for which we were able to map the exact breakpoints. We also identified four known mutations - three in ASPM and one in WDR62. The latter was initially deemed to be a missense mutation but we demonstrate here that it affects splicing. As to ASPM, as many as 17 out of 27 MCPH5 families that we ascertained in our sample were found to carry the previously reported founder mutation p.Trp1326*. This study adds to the mutational spectra of four known MCPH-associated genes and updates our knowledge about the genetic heterogeneity of MCPH in the Pakistani population considering its ethnic diversity.

Early vs delayed closure of loop defunctioning ileostomy in patients undergoing distal colorectal resections: an integrated systematic review and meta-analysis of published randomized controlled trials.

AIMS: The objective of this study was to evaluate the surgical outcomes and feasibility of early loop defunctioning ileostomy closure, within 2 weeks of index surgery, in patients undergoing distal colorectal resection. METHODS: A systematic review of the literature on published randomized controlled trials reporting the feasibility and outcomes on early vs delayed closure of loop defunctioning ileostomy in patients undergoing distal colorectal resection using the principles of meta-analysis on RevMan 5.4 statistical software was undertaken. RESULTS: Four randomized, controlled trials on 446 patients evaluating the feasibility and outcomes on early vs delayed closure of loop defunctioning ileostomy in patients undergoing low colorectal resection were analysed. There were 176 patients in the early closure group and 270 patients in the delayed closure group. The risk of anastomotic leak [risk ratio 0.37 (CI: 0.10-1.42), P = 0.15], anastomotic stenosis [risk ratio 4.79 (CI: 0.23-98.47), P = 0.31] and postoperative complications [risk ratio 0.75 (CI: 0.48-1.16), P = 0.19] was similar in both groups. In addition, there was no significant difference between the groups with regard to the duration of operation [standardized mean difference -0.49 (CI: -01.09, -0.12), P = 0.12] and length of hospitalization [standardized mean difference -0.04 (CI: -0.25, -0.18), P = 0.75]. CONCLUSIONS: Early closure of loop defunctioning ileostomy in patients undergoing distal colorectal resection is feasible with comparable outcomes to delayed closure.

Stressors affecting nursing students in Pakistan.

AIM: To determine factors contributing to stress experienced by preregistration nursing students in Pakistan, using the Stressors in Nursing Students scale. The aim was to explore the psychometric properties of this instrument and to investigate the effect of a range of demographic variables on the perception of stressors in nursing students. BACKGROUND: Nursing is a stressful profession, and nursing students may experience more stress due to competing demands and challenges of nursing education, assessment, placements and worries about employment prospects. METHODS: In this cross-sectional survey, data from 726 nursing students from 11 schools of nursing in Karachi, Pakistan, were collected using a questionnaire. Data were analysed using descriptive as well inferential statistics. An exploratory factor analysis was also conducted. RESULTS: There was no apparent factor structure to the Stressors in Nursing Students scale, unlike in previous studies. The total score on the Stressors in Nursing Students scale was related to gender with males scoring higher. The score generally increased over 4 years of the programme, and students in private schools of nursing scored higher than those in public schools of nursing. CONCLUSION: Nursing students in Pakistan do not appear to differentiate between different stressors, and this may be due to cultural differences in the students and to the structure of the programme and the articulation between the academic and clinical aspects. Likewise, cultural reasons may account for differences between stress experienced by male and female students. IMPLICATIONS FOR NURSING AND POLICY: The fact that scores on the Stressors in Nursing Students scale increased over 4 years of the programme and males scored higher than females should alert nursing schools and policymakers related to nursing education and workforce to pay attention to prevent attrition from nursing programmes.

Combining 3D human in vitro methods for a 3Rs evaluation of novel titanium surfaces in orthopaedic applications.

In this study, we report on a group of complementary human osteoblast in vitro test methods for the preclinical evaluation of 3D porous titanium surfaces. The surfaces were prepared by additive manufacturing (electron beam melting [EBM]) and plasma spraying, allowing the creation of complex lattice surface geometries. Physical properties of the surfaces were characterized by SEM and profilometry and 3D in vitro cell culture using human osteoblasts. Primary human osteoblast cells were found to elicit greater differences between titanium sample surfaces than an MG63 osteoblast-like cell line, particularly in terms of cell survival. Surface morphology was associated with higher osteoblast metabolic activity and mineralization on rougher titanium plasma spray coated surfaces than smoother surfaces. Differences in osteoblast survival and metabolic activity on titanium lattice structures were also found, despite analogous surface morphology at the cellular level. 3D confocal microscopy identified osteoblast organization within complex titanium surface geometries, adhesion, spreading, and alignment to the biomaterial strut geometries. Mineralized nodule formation throughout the lattice structures was also observed, and indicative of early markers of bone in-growth on such materials. Testing methods such as those presented are not traditionally considered by medical device manufacturers, but we suggest have value as an increasingly vital tool in efficiently translating pre-clinical studies, especially in balance with current regulatory practice, commercial demands, the 3Rs, and the relative merits of in vitro and in vivo studies. Biotechnol. Bioeng. 2016;113: 1586-1599. © 2015 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Factors associated with failure to achieve a glycated haemoglobin target of <8.0% in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial.

The aim of this study was to identify the clinical features of participants in the standard therapy arm of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) glycaemia trial who failed to reach the glycated haemoglobin (HbA1c) target. We analysed 4685 participants in the standard therapy arm, comparing participants who reached the HbA1c target of <8.0% with those whose HbA1c level was ≥8.0% 12 months after randomization. Baseline and 12-month clinical characteristics were compared. At 12 months after randomization, 3194 participants had HbA1c <8.0% and 1491 had HbA1c ≥8.0%. Black race [odds ratio (OR) 0.74, 95% confidence interval (CI) 0.61-0.89; p = 0.002], severe hypoglycaemia (OR 0.57, CI 0.37-0.89; p = 0.014) and insulin use (OR 0.51, CI 0.40-0.65; p < 0.001) were associated with failure to reach HbA1c goal at 12 months in the adjusted model. Even with free medications, free visits with clinicians and aggressive titration of medications, >30% of participants in the standard arm of the ACCORD trial had an HbA1c ≥8.0% at 1 year. Participants who were black, had severe hypoglycaemia and were on insulin were more likely to have an above-target HbA1c concentration after 12 months on the standard protocol.

Molecular characterization of Staphylococcus aureus isolates causing skin and soft tissue infections in patients from Malakand, Pakistan.

Comparatively few studies have been published describing Staphylococcus aureus/MRSA epidemiology in Central Asia including Pakistan. Here, we report the genotyping of Staphylococcus aureus strains (that include both methicillin-susceptible and methicillin-resistant Staphylococcus aureus) from community- and hospital-acquired skin and soft-tissue infections in a tertiary care hospital in the Malakand district of the Khyber Pakhtunkhwa Province of Pakistan. Forty-five isolates of Staphylococcus aureus were characterized by microarray hybridization. Twenty isolates (44 %) were MRSA, whereas 22 (49 %) were PVL-positive. Fourteen isolates (31 %) harboured both mecA and PVL genes. The dominant clones were CC121-MSSA (n = 15, 33 %) and the PVL-positive "Bengal Bay Clone" (ST772-MRSA-V; n = 13, 29 %). The PVL-positive CC8-MRSA-IV strain "USA300" was found once. The pandemic ST239-MRSA-III strain was absent, although it has previously been observed in Pakistan. These observations require a re-assessment of schemes for initial antibiotic therapy to cover MRSA and they emphasise the need for a rapid and non-molecular test for PVL.

Phase II study results of a replacement therapy for hereditary angioedema with subcutaneous C1-inhibitor concentrate.

BACKGROUND: Hereditary angioedema (HAE) due to C1 inhibitor deficiency manifests as recurrent swelling attacks that can be disabling and sometimes fatal. Long-term prophylaxis with twice-weekly intravenous injections of plasma-derived C1-inhibitor (pdC1-INH) has been established as an effective treatment. Subcutaneous (SC) administration of pdC1-INH has not been studied in patients with HAE. METHODS: This open-label, dose-ranging, crossover study (COMPACT Phase II) was conducted in 18 patients with type I or II HAE who received two of twice-weekly 1500, 3000, or 6000 IU SC doses of highly concentrated volume-reduced CSL830 for 4 weeks each. The mean trough plasma levels of C1-INH functional activity, C1-INH and C4 antigen levels during Week 4, and overall safety and tolerability were evaluated. The primary outcome was model-derived steady-state trough C1-INH functional activity. RESULTS: After SC CSL830 administration, a dose-dependent increase in trough functional C1-INH activity was observed. C1-INH and C4 levels both increased. The two highest dose groups (3000 and 6000 IU) achieved constant C1-INH activity levels above 40% values, a threshold that was assumed to provide clinical protection against angioedema attacks. Compared with intravenous injection, pdC1-INH SC injection with CSL830 showed a lower peak-to-trough ratio and more consistent exposures. All doses were well tolerated. Mild-to-moderate local site reactions were noted with pain and swelling being the most common adverse event. CONCLUSIONS: Subcutaneous volume-reduced CSL830 was well tolerated and led to a dose-dependent increase in physiologically relevant functional C1-INH plasma levels. A clinical outcome study of SC CSL830 in patients with HAE warrants further investigation.

Oncological feasibility of laparoscopic distal pancreatectomy for adenocarcinoma: a single-institution comparative study.

BACKGROUND: Laparoscopic distal pancreatectomy (LDP) is performed increasingly for pancreatic pathology in the body and tail of the pancreas. However, only few reports have compared its oncological efficacy with open distal pancreatectomy (ODP). We compared these two techniques in patients with pancreatic ductal adenocarcinoma. METHODS: From a prospectively maintained database, all patients who underwent either LDP or ODP for adenocarcinoma in the body and tail of the pancreas between January 2008 and December 2011 were compared. Data were analysed using SPSS(®) v19 utilising standard tests. A p value <0.05 was considered significant. RESULTS: Of 101 patients who underwent distal pancreatectomy, 22 had histologically confirmed adenocarcinoma (LDP n = 8, ODP n = 14). Both groups were well matched for age and the size of tumour (22 vs. 32 mm, p = 0.22). Intraoperative blood loss was 306 ml compared with 650 ml for ODP (p = 0.152). A longer operative time was noted for LDP (376 vs. 274 min, p < 0.05). Total length of stay was shorter for LDP compared with ODP (8 vs. 12 days, p = 0.05). The number of postoperative pancreatic fistulas were similar (LDP n = 2 vs. ODP n = 3, p = 0.5). Complete resection (R0) was achieved in 88 % of LDP (n = 7) compared with 86 % of ODP (n = 12). The median number of lymph nodes harvested was 16 for LDP versus 14 for ODP. Overall 3-year survival also was similar: LDP = 82 %, ODP = 74 % (p = 0.89). CONCLUSIONS: From an oncological perspective, LDP is a viable procedure and its results are comparable to ODP for ductal adenocarcinomas arising in the body and tail of the pancreas.

Sensitizing undergraduate medical students to consultation skills: A pilot study.

BACKGROUND: Good consultation skills help physicians to diagnose the problems of the patient more accurately, and foster a therapeutic relationship. We describe a pilot study that used role-play with peers as a method to sensitize first clinical year medical students to consultation skills Methods. Students were divided into groups of three where one acted as a doctor, the second as a patient and the third as an observer. Students were asked to perform a role-play of a prepared clinical scenario where the patient had a hidden fear of malignancy. Observations were recorded in a simplified Calgary-Cambridge consultation checklist. Students' feedback and their emotions written after the role-play were analysed and discussed. Assessment of their learning was done with an objective structured clinical examination. RESULTS: Students' feedback revealed that they were sensitized to the importance of starting the consultation with an open question, listening to the opening statement, non-verbal.

Cracking the Whippet: The Inconsistent Treatment of Myeloneuropathy Secondary to Chronic Nitrous Oxide Misuse.

Background The recreational abuse of nitrous oxide (N2O) is becoming increasingly prevalent within the United Kingdom and across the globe. Chronic abuse can cause nerve and spinal cord damage through the functional inactivation of vitamin B12. We present six cases from a single centre in the United Kingdom between 2016 and 2020 with neurological complications from N2O abuse, ranging from paraesthesia to subacute combined degeneration of the spinal cord. Methodology A retrospective review of all neurology admissions to Queen Elizabeth Hospital Birmingham (QEHB) between 2016 and 2020 was conducted to identify patients admitted with a diagnosis of neurological dysfunction (neuropathy or myelopathy) in association with chronic N2O misuse. The Prescribing Information and Communication System was used to collect data on demographics, reported N2O misuse, presenting symptoms, results of blood tests, nerve conduction studies and treatment regimens. All patients gave verbal consent for inclusion in the case series. Data were anonymised and analysed by the corresponding author. Results All patients were males aged between 22 and 28 years. In all cases, the patients were admitted with the abuse of N2O whippet canisters (ranging from 20 to 500 canisters per session), presenting with a combination of sensory and motor disturbance. Clinical suspicion, in the context of a history of N2O abuse, along with elevated blood concentrations of methylmalonic acid (MMA) and homocysteine, and nerve conduction studies, was the cornerstone of the diagnosis. All patients were treated with parenteral vitamin B12, though individual regimens differed, with no standardisation in the duration or frequency of treatment. All patients received intramuscular (IM) vitamin B12 injections during admission, with one patient receiving oral vitamin B12 before being switched to IM vitamin B12 injections. One patient received additional folic acid as a treatment adjunct. Prescriptions were most varied on discharge with huge discrepancies in duration and frequency of vitamin B12 replacement, ranging from no B12 replacement at all to IM injections once weekly for eight weeks.  Conclusions The variability in route, dose and duration of vitamin B12 treatment, along with the variable use of adjunctive therapy reported in the literature, highlights the current lack of consensus in managing N2O neurotoxicity.

Real-world comparison of terlipressin vs. octreotide as an adjuvant treatment in the management of variceal bleeding.

Variceal bleeding is a major complication and the leading cause of death in patients with cirrhosis and portal hypertension. This study aims to compare the efficacy and safety of terlipressin vs octreotide as an adjuvant to endoscopic management of patients with esophageal variceal bleeding in a real-time scenario. We reviewed the medical records of patients with esophageal variceal bleeding from January 2005 to December 2020 at our tertiary care Aga Khan University Hospital. Mortality was assessed after 6 weeks. A total of 842 patients with variceal bleed were evaluated. 624 patients (74.1%) and 218 patients (25.9%) received Terlipressin and Octreotide respectively. On multiple regression analysis, cardiac events during hospital stay (OR: 11.22), presence of Porto-systemic encephalopathy (OR: 3.79), and elevated bilirubin levels at the time of presentation were found to be independent risk factors for increased six weeks mortality. Moreover, cardiac events during hospital stay (OR: 3.26), Porto-systemic encephalopathy at presentation (OR: 3.06), and octreotide administration (OR: 1.80) were identified as independent risk factors for increased length of hospital stay. Terlipressin and Octreotide have similar outcomes in terms of control of bleeding, hospital stay, mortality, and side effects when used as adjuvant therapy for the management of variceal bleeding.

Catalyst driven optical properties of the self-assembled ZnS nanostructures.

In this paper, we present the effect of different catalysts (Mn, Au and Sn) on the room temperature photoluminescence and Raman spectra of VLS grown ZnS nanostructures. The catalysts found to self-dope the ZnS nanostructures during its growth were confirmed by XRD and XPS results. The extent of self-doping, which depended on the type and size of catalysts, was observed strongly to have affected the optical properties of ZnS nanostructures, particularly intrinsic defects like S and Zn vacancies. Intense broad bands in the visible due to intrinsic defects, namely Zn and S vacancies were observed, which were quite different for each catalyst as well as for the size of the catalyst. Au and Mn catalyzed ZnS nanostructures also showed creation of catalyst related defects, which were absent in the case of Sn. From the PL spectra, an estimation of the Zn and S vacancies was made for each type of catalyzed ZnS nanostructures. Surface optic (SO) phonon modes of the ZnS nanostructures were also observed to behave differently for the three catalysts. The dielectric continuum model was applied to determine the correlation length and variation in the surface potential modulations in these nanostructures. It was deduced from optical studies that the location of dopant in self-doped ZnS nanostructures strongly affected the luminescence properties. Finally it was concluded that self-doping using a suitable catalyst can be a simple and controllable way to dope ZnS nanostructures with tailored optical characteristics.

Prognostic factors and survival after resection of colorectal liver metastasis in the era of preoperative chemotherapy: an 11-year single-centre study.

INTRODUCTION: A variety of factors have been identified in the literature which influence survival following resection of colorectal liver metastases (CRLM). Much of this literature is historical, and its relevance to contemporary practice is not known. The aim of this study was to identify those factors which influence survival during the era of preoperative chemotherapy in patients undergoing resection of CRLM in a UK centre. METHODS: All patients having liver resection for CRLM during an 11-year period up to 2011 were identified from a prospectively maintained database. Prognostic factors analysed included tumour size (≥5 or <5 cm), lymph node status of the primary tumour, margin positivity (R1; <1 mm), neo-adjuvant chemotherapy (for liver), tumour differentiation, number of liver metastases (≥4), preoperative carcinoembryonic antigen (CEA; ≥200 ng/ml) and whether metastases were synchronous (i.e. diagnosed within 12 months of colorectal resection) or metachronous to the primary tumour. Overall survival (OS) was compared using Kaplan-Meier plots and a log rank test for significance. Multivariate analysis was performed using a Cox regression model. Statistical analysis was performed in SPSS v19, and p < 0.05 was considered to be significant. RESULTS: 432 patients underwent resection of CRLM during this period (67% male; mean age 64.5 years), and of these, 54 (13.5%) had re-resections. The overall 5-year survival in this series was 43% with an actuarial 10-year survival of 40%. A preoperative CEA ≥200 ng/ml was present in 10% of patients and was associated with a poorer 5-year OS (24 vs. 45%; p < 0.001). A positive resection margin <1 mm was present in 16% of patients, and this had a negative impact on 5-year OS (15 vs. 47%; p < 0.001). Tumour differentiation, number, biliary or vascular invasion, size, relationship to primary disease, nodal status of the primary disease or the use of neo-adjuvant chemotherapy had no impact on OS. Multivariate analysis identified only the presence of a positive resection margin (OR 1.75; p < 0.05) and a preoperative CEA ≥200 ng/ml (OR 1.88; p < 0.01) as independent predictors of poor OS. CONCLUSION: Despite the wide variety of prognostic factors reported in the literature, this study was only able to identify a preoperative CEA ≥200 ng/ml and the presence of tumour within 1 mm of the resection margin as being of value in predicting survival. These variables are likely to identify patients who may benefit from intensive follow-up to enable early aggressive treatment of recurrent disease.

Descriptive sensory profile of cow and buffalo milk Cheddar cheese prepared using indigenous cultures.

The objective of the study was to compare the sensory profile of Cheddar cheese prepared from cow and buffalo milk using indigenous and commercial cultures. Commercially available and locally isolated, indigenous starter cultures were used to prepare cow and buffalo milk Cheddar cheese. The cheese was ripened at 4 and 12°C and analyzed for descriptive sensory profile by a panel of 10 assessors after 60 and 120 d of ripening. On evaluation, the mean scores for odor, flavor, and texture attributes obtained for buffalo milk cheese were significantly higher than those obtained for cow milk cheese. For most of the traits, cheese samples prepared from indigenous cultures and ripened at higher temperature received higher descriptive scores compared with those of commercial cultures and ripened at lower degrees. Milk sources highly significantly affected the "creamy" and "sour" traits of odor; the "creamy," "smoky," and "soapy" flavors; and all the texture attributes except "maturity." Starter cultures considerably influenced the production of "acidic," "bitter," "sweet," and "sour" characteristics. The use of elevated ripening temperature showed noticeable effect on all the characteristics except the "creamy" odor and flavor. Principal component analysis and hierarchical cluster analysis also showed that milk sources, starter cultures, and ripening temperatures significantly influenced the sensory characteristics.

First Report of Citrus tristeza virus in National Germplasm of Citrus in Afghanistan.

Rejuvenation of the horticulture industry is a government priority in Afghanistan. With that purpose, European Commission-supported programs specifically focus on greater access to improved and appropriate planting materials to increase the quantity and quality of more competitive horticultural products. Establishment of a biotechnology laboratory was considered essential support to horticulture sector development. This laboratory has begun screening the health status of the Afghan Germplasm National Collection to ensure multiplication of not only the best selected varieties or ecotypes but also to avoid reproduction and distribution of virus-infected fruit trees. Symptom inspection and sample collection for viral diseases was carried out in the citrus orchard during survey activity at the National Collection Experimental Farm in Jalalabad (Nangarhar Province). Ninety-nine variety plots (one row of five plants) were inspected visually and samples from two plants for each plot were collected and analyzed by double-antibody sandwich (DAS)-ELISA. Plants showing vein flecking, yellowing, and plant decline symptoms were observed in several plots. Four accessions were found to be infected by Citrus tristeza virus (CTV): kumquat cv. Margarita (isolates J4 and J8), orange cv. Mahali (J61), mandarin group cv. Fruter (J76), and rough lemon cv. Mahali (J101). Identified isolates have been characterized molecularly. A 655-nt fragment, corresponding to the major coat protein gene, has been amplified from all ELISA-positive samples by reverse transcription (RT)-PCR using CTVF (5'-TAATGGACGACGAACAAAGA-3') and CTVR (5'-CCAAGCTGCCTGACATTAGT-3') primers. Sequence analysis revealed high similarity, ranging from 91.1 to 99.8%, within CTV isolates detected in Jalalabad. In accordance with the phylogenetic groups previously defined (page 8 in: Proceedings of the 15th Conference of the International Organization of Citrus Virologists, 2002), nucleotide sequences of Afghan CTV isolates investigated in the current work cluster in Group 1 (J4 and J8), Group 4 (J61 and J76), and Group 5 (J101). In particular, J4 and J8 isolates show, respectively, identity of 99.4 and 99.2% with reference isolate T36 (GenBank Accession No. M76485) from the United States (Florida). Moreover, in Group 4, isolate J61 and J76 were more similar to ANO-1 isolate (GenBank Accession No. DQ211658) from Egypt (identity of 98.5 and 98.0%, respectively) than to isolate 443-4 (GenBank Accession No. AY791844) from Croatia (97.4 and 97.5%, respectively). Finally, isolate J101 in Group 5, shows identity of 95.6% with isolates C268-2 (GenBank Accession No. AY750770) and C269-6 (GenBank Accession No. AY750775) from Argentina. To our knowledge, our results identified for the first time CTV-infected plants in Afghanistan. The presence of CTV in four accessions of the national citrus collection is of concern for Afghan horticulture. Implementation of the certification schemes is therefore necessary to guarantee the production and the employment of virus-free propagating material.

Nutritional evaluation of distillery sludge and its effect as a substitute of canola meal on performance of broiler chickens.

The study was conducted to investigate the chemical composition of distillery yeast sludge and its inclusion in broiler diets to replace canola meal. Raw distillery yeast sludge was washed with water using water and sludge in the ratio 6:1, respectively. Proximate analysis of raw distillery yeast sludge and washed distillery sludge was carried out for crude protein (CP), true protein (TP), ether extract (EE), ash, acid insoluble ash and nitrogen free extract (NFE) determination. Mineral contents and amino acid profile of raw distillery yeast sludge and washed distillery sludge were also determined. After chemical evaluation, four iso-caloric and iso-nitrogenous broiler starter and finisher diets were prepared in mash form using 0 (control), 4, 8 and 12% levels of washed distillery sludge replacing canola meal. One hundred and twenty day-old broiler chicks were randomly distributed into 12 experimental units in such a way that each diet was offered to three experimental units, each comprising of 10 chicks. It was observed that washing affected the nutrients either by decreasing or increasing their concentration. It decreased the total mineral contents whereas CP, TP, EE and NFE contents increased. Washing also increased amino acid profile. Average feed intake and weight gain were higher in birds fed diet containing 8% washed distillery sludge and lower in birds fed diet containing 0% washed distillery sludge. Feed cost per kg live weight gain decreased significantly as the level of washed distillery sludge was increased in the diet. Average heart, liver and pancreas weights decreased with increased level of washed distillery sludge in the diet. The study revealed that after washing, distillery yeast sludge can be used successfully in broiler diets up to the level of 8% without any adverse effect on broiler's performance.