Low androgen signaling rescues genome integrity with innate immune response by reducing fertility in humans.

Development of the gonads under complex androgen regulation is critical for germ cells specification. In this work we addressed the relationship between androgens and genomic integrity determining human fertility. We used different study groups: individuals with Differences of Sex Development (DSD), including Complete Androgen Insensitivity Syndrome (CAIS) due to mutated androgen receptor (AR), and men with idiopathic nonobstructive azoospermia. Both showed genome integrity status influenced by androgen signaling via innate immune response activation in blood and gonads. Whole proteome analysis connected low AR to interleukin-specific gene expression, while compromised genome stability and tumorigenesis were also supported by interferons. AR expression was associated with predominant DNA damage phenotype, that eliminated AR-positive Sertoli cells as the degeneration of gonads increased. Low AR contributed to resistance from the inhibition of DNA repair in primary leukocytes. Downregulation of androgen promoted apoptosis and specific innate immune response with higher susceptibility in cells carrying genomic instability.

Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

AZFa Y gene, DDX3Y, evolved novel testis transcript variants in primates with proximal 3´UTR polyadenylation for germ cell specific translation.

Translational control is a major level of gene expression regulation in the male germ line. DDX3Y located in the AZFa region of the human Y chromosome encodes a conserved RNA helicase important for translational control at the G1-S phase of the cell cycle. In human, DDX3Y protein is expressed only in premeiotic male germ cells. In primates, DDX3Y evolved a second promoter producing novel testis-specific transcripts. Here, we show primate species-specific use of alternative polyadenylation (APA) sites for these testis-specific DDX3Y transcript variants. They have evolved subsequently in the 3´UTRs of the primates´ DDX3Y transcripts. Whereas a distal APA site (PAS4) is still used for polyadenylation of most DDX3Y testis transcripts in Callithrix jacchus; two proximal APAs (PAS1; PAS2) are used predominantly in Macaca mulatta, in Pan trogloydates and in human. This shift corresponds with a significant increase of DDX3Y protein expression in the macaque testis tissue. In chimpanzee and human, shift to predominant use of the most proximal APA site (PAS1) is associated with translation of these DDX3Y transcripts in only premeiotic male germ cells. We therefore assume evolution of a positive selection process for functional DDX3Y testis transcripts in these primates which increase their stability and translation efficiency to promote its cell cycle balancing function in the human male germ line.

The challenge of comparing digitally captured signatures registered with different software and hardware.

Along with the growing popularity of electronic documents authorised with digitally captured signatures, such evidence has appeared in the work of forensic practitioners. Many different vendors offer signature pads with varying specifications. It is therefore expected that forensic handwriting experts will be called upon to compare questioned and known samples captured with completely or partially different hardware and software combinations. Such cases may be challenging as numerical handwriting data produced by various equipment may differ not only in the type of information captured and its quality, but also in its structure and coding. In this research, numerical data of handwriting - i.e. spatial coordinates, force, and time values - were acquired with 26 different combinations of hardware and software to study characteristics of their coding. The analysis of samples revealed that scaling of numerical data is not only hardware but also software dependent. Therefore, their compliance with the ISO/IEC 19794-7 standard is recommended to improve the data interoperability. This standard emphasizes the importance of supplementing numerical signature data with scaling ratios of the used signing solution. The paper also includes descriptions of several phenomena observed in the acquired data to highlight possible pitfalls in performing inter-solution comparisons in casework.

Human AZFb deletions cause distinct testicular pathologies depending on their extensions in Yq11 and the Y haplogroup: new cases and review of literature.

Genomic AZFb deletions in Yq11 coined "classical" (i.e. length of Y DNA deletion: 6.23 Mb) are associated with meiotic arrest (MA) of patient spermatogenesis, i.e., absence of any postmeiotic germ cells. These AZFb deletions are caused by non-allelic homologous recombination (NAHR) events between identical sequence blocks located in the proximal arm of the P5 palindrome and within P1.2, a 92 kb long sequence block located in the P1 palindrome structure of AZFc in Yq11. This large genomic Y region includes deletion of 6 protein encoding Y genes, EIFA1Y, HSFY, PRY, RBMY1, RPS4Y, SMCY. Additionally, one copy of CDY2 and XKRY located in the proximal P5 palindrome and one copy of BPY1, two copies of DAZ located in the P2 palindrome, and one copy of CDY1 located proximal to P1.2 are included within this AZFb microdeletion. It overlaps thus distally along 2.3 Mb with the proximal part of the genomic AZFc deletion. However, AZFb deletions have been also reported with distinct break sites in the proximal and/or distal AZFb breakpoint intervals on the Y chromosome of infertile men. These so called "non-classical" AZFb deletions are associated with variable testicular pathologies, including meiotic arrest, cryptozoospermia, severe oligozoospermia, or oligoasthenoteratozoospermia (OAT syndrome), respectively. This raised the question whether there are any specific length(s) of the AZFb deletion interval along Yq11 required to cause meiotic arrest of the patient's spermatogenesis, respectively, whether there is any single AZFb Y gene deletion also able to cause this "classical" AZFb testicular pathology? Review of the literature and more cases with "classical" and "non-classical" AZFb deletions analysed in our lab since the last 20 years suggests that the composition of the genomic Y sequence in AZFb is variable in men with distinct Y haplogroups especially in the distal AZFb region overlapping with the proximal AZFc deletion interval and that its extension can be "polymorphic" in the P3 palindrome. That means this AZFb subinterval can be rearranged or deleted also on the Y chromosome of fertile men. Any AZFb deletion observed in infertile men with azoospermia should therefore be confirmed as "de novo" mutation event, i.e., not present on the Y chromosome of the patient's father or fertile brother before it is considered as causative agent for man's infertility. Moreover, its molecular length in Yq11 should be comparable to that of the "classical" AZFb deletion, before meiotic arrest is prognosed as the patient's testicular pathology.

Features of digitally captured signatures vs. pen and paper signatures: Similar or completely different?

The question of whether digitally captured signatures and conventional signatures executed with a pen on paper differ in their characteristics is of practical relevance for forensic handwriting examiners. Due to gaps in the current literature, the present research is dedicated to this issue. Eighty persons signed in three conditions: a) with a stylus on a pad, b) with an inking pen on a sticky note attached to a signature pad allowing to obtain a digital and an analogue version on paper of one and the same writing simultaneously, and c) with a pen on paper. The first step was to investigate to what extent the character shape and number of pen lifts differ between the digital and analogue representation of one and the same signature. This revealed minor differences which are due to technical characteristics of the devices used. The observed distortions are of minor practical relevance according to ratings by eight participating forensic handwriting examiners. Subsequently, signature characteristics were compared between the three different writing conditions in a casework-oriented way. Statistical multi-level models indicate significant differences between the three signature types, but minor effect sizes in most of the examined characteristics. From the point of view of the participating handwriting examiners, these factors do not fundamentally restrict the comparability between digitally captured and conventional signatures in practice. However, caution should be exercised when generalising the results, as several factors, such as the usage of different signature pads as well as signatures made with the finger instead of a stylus, could result in more important differences compared to pen and paper signatures.

Gonadoblastoma Y locus genes expressed in germ cells of individuals with dysgenetic gonads and a Y chromosome in their karyotypes include DDX3Y and TSPY.

STUDY QUESTION: Which Y genes mapped to the 'Gonadoblastoma Y (GBY)' locus on human Y chromosome are expressed in germ cells of individuals with some Differences of Sexual Development (DSD) and a Y chromosome in their karyotype (DSD-XY groups)? SUMMARY ANSWER: The GBY candidate genes DDX3Y and TSPY are expressed in the germ cells of DSD-XY patients from distinct etiologies: patients with mixed gonadal dysgenesis (MGD) and sex chromosome mosaics (45,X0/46,XY; 46,XX/46,XY); patients with complete androgen insensitivity (CAIS), patients with complete gonadal dysgenesis (CGD; e.g. Swyer syndrome). WHAT IS KNOWN ALREADY: A GBY locus was proposed to be present on the human Y chromosome because only DSD patients with a Y chromosome in their karyotype have a high-although variable-risk (up to 55%) for germ cell tumour development. GBY was mapped to the proximal part of the short and long Y arm. TSPY located in the proximal part of the short Y arm (Yp11.1) was found to be a strong GBY candidate gene. It is expressed in the germ cells of DSD-XY patients with distinct etiologies but also in foetal and pre-meiotic male spermatogonia. However, the GBY region extends to proximal Yq11 and therefore includes probably more than one candidate gene. STUDY DESIGN, SIZE, DURATION: Protein expression of the putative GBY candidate gene in proximal Yq11, DDX3Y, is compared with that of TSPY in serial gonadal tissue sections of 40 DSD-XY individuals from the three DSD patient groups (MGD, Complete Androgen Insensitivity Syndrome [CAIS], CGD) with and without displaying malignancy. Expression of OCT3/4 in the same tissue samples marks the rate of pluripotent germ cells. PARTICIPANTS/MATERIALS, SETTING, METHOD: A total of 145 DSD individuals were analysed for the Y chromosome to select the DSD-XY subgroup. PCR multiplex assays with Y gene specific marker set score for putative microdeletions in GBY Locus. Immunohistochemical experiments with specific antisera mark expression of the GBY candidate proteins, DDX3Y, TSPY, in serial sections of the gonadal tissue samples; OCT3/4 expression analyses in parallel reveal the pluripotent germ cell fraction. MAIN RESULTS AND THE ROLE OF CHANCE: Similar DDX3Y and TSPY protein expression patterns were found in the germ cells of DSD-XY patients from each subgroup, independent of age. In CAIS patients OCT3/4 expression was often found only in a fraction of these germ cells. This suggest that GBY candidate proteins are also expressed in the non-malignant germ cells of DSD-XY individuals like in male spermatogonia. LIMITATIONS, REASONS FOR CAUTION: Variation of the expression profiles of GBY candidate genes in the germ cells of some DSD-XY individuals suggests distinct transcriptional and translational control mechanisms which are functioning during expression of these Y genes in the DSD-XY germ cells. Their proposed GBY tumour susceptibility function to transform these germ cells to pre-malignant GB/Germ Cell Neoplasia in Situ (GB/GCNIS) cells seems therefore to be limited and depending on their state of pluripotency. WIDER IMPLICATIONS OF THE FINDINGS: These experimental findings are of general importance for each individual identified in the clinic with DSD and a Y chromosome in the karyotype. To judge their risk of germ cell tumour development, OCT3/4 expression analyses on their gonadal tissue section is mandatory to reveal the fraction of germ cells still being pluripotent. Comparative expression analysis of the GBY candidate genes can be helpful to reveal the fraction of germ cells with genetically still activated Y chromosomes contributing to further development of malignancy if at high expression level. STUDY FUNDING/COMPETING INTEREST(S): This research project was supported by a grant (01GM0627) from the BMBF (Bundesministerium für Bildung und Forschung), Germany to P.H.V. and B.B. The authors have no competing interests.

[Perioperative safety of intraperitoneal aerosol chemotherapy : Analysis of our first 111 pressurized intraperitoneal aerosol chemotherapy (PIPAC) procedures]. / Perioperative Sicherheit der intraperitonealen Aerosolchemotherapie : Analyse unserer ersten 111 PIPAC("pressurized intraperitoneal aerosol chemotherapy")-Prozeduren.

BACKGROUND: Pressurized intraperitoneal aerosol chemotherapy (PIPAC) is a new tool in the treatment of patients with peritoneal carcinomatosis. The aerosol containing chemotherapeutic drugs is administered laparoscopically into the abdominal cavity to achieve a local treatment effect. This can be carried out in combination with systemic chemotherapy. MATERIAL AND METHODS: Within the framework of a register study, we prospectively documented and evaluated the data of our first 111 PIPAC procedures. The analysis focused on perioperative patient safety and safety at the workplace. Perioperative clinical patient data were analyzed and the platinum concentration in the operating room was checked by wipe samples. RESULTS: A total of 62 patients were scheduled for PIPAC and 121 operations were carried out. In 9 procedures a secure access to the abdomen could not be found and 54 patients received 111 PIPAC treatments. One patient died as a result of intestinal perforation, six bowel lesions were treated immediately and healed without further complications. A further patient developed a postoperative renal failure. Otherwise, there was no major complications and no cases of toxicity. CONCLUSION: The PIPAC procedure can be used as a supplement to systemic drug treatment for peritoneal carcinomatosis. An exact selection of suitable patients is important. The PIPAC is a low-risk procedure when performed under strict inclusion criteria and under standardized conditions, for the patients and also the surgical staff.

Postprandial glucose and insulin response to a high-fiber muffin top containing resistant starch type 4 in healthy adults: a double-blind, randomized, controlled trial.

OBJECTIVES: VERSAFIBE™ 2470 resistant starch (RS) is an RS type 4 that is derived from high-amylose maize starch,70% total dietary fiber (TDF; AOAC method 2009.01). This was a randomized, double-blind, crossover study to evaluate the postprandial blood glucose and insulin responses of healthy adults (n = 28) after the consumption of a muffin top made with VERSAFIBE™ 2470 RS (11.6 g TDF fiber muffin top) or a control muffin top (0.9 g TDF). METHODS: The muffin tops were matched for weight, total carbohydrate, sugars, protein, and fat. During each treatment period, subjects consumed a standard evening meal, fasted for 12 h, and arrived at the study clinic the following morning. Serum glucose, serum insulin, and capillary glucose were measured at 0, 15, 30, 45, 60, 90, and 120 min after muffin top consumption. The subjects completed a 7-d washout period between treatments. RESULTS: The consumption of the fiber muffin top resulted in a significant 33% reduction in postprandial serum glucose incremental area under the curve from 0 to 120 min and an 8% decrease in maximum glucose concentration versus the control muffin (P = 0.037 and P = 0.007, respectively). The fiber muffin top reduced postprandial serum insulin incremental area under the curve from 0 to 120 min by 38% compared with the control muffin top (P <0.001), which aligns with the blood glucose data. CONCLUSIONS: This study demonstrated that the inclusion of a practical dose of dietary fiber (11.6 g TDF) from VERSAFIBE™ 2470 RS in a bakery product significantly reduced postprandial glucose and insulin responses in healthy adults.

Gastrointestinal Tolerance and Glycemic Response of Isomaltooligosaccharides in Healthy Adults.

Ingredients delivering functional and nutritional benefits are of interest to food manufacturers. Isomaltooligosaccharides (IMOs) which serve as alternate sweeteners fit into this category. IMOs are a mixture of α-(1 → 6) and α-(1 → 4)-linked glucose oligomers, synthesized by an enzymatic reaction from starch (corn, tapioca). The aim of this study was to evaluate the fermentability and glycemic response of IMO in a healthy population. Two randomized, double-blind, placebo-controlled, cross-over human studies were conducted. In the first study (n = 26), participants' breath hydrogen over 24 h, gastrointestinal tolerance, and glycemic and insulinemic response to BIOLIGOTM IL5040 isomaltooligosaccharide were measured. In another study (n = 10), participants' two-hour post-prandial glycemic response to BIOLIGOTM IL5040 isomaltooligosaccharide and BIOLIGOTM IL7010 isomaltooligosaccharide was measured compared to dextrose (control). The IMOs differed in the composition of mono and di-saccharide sugars. IMO syrup dose was matched for 50 g of total carbohydrates and was consumed by mixing in water (237 mL/8 oz.). Mean composite gastrointestinal score was not significantly different (p = 0.322) between the control (1.42) and IMO (1.38). Lack of difference in glycemic response (p = 0.662), with no impact on breath hydrogen (24 h; p = 0.319) and intestinal tolerance, demonstrates that IMO is digestible and can be used to replace sugars in product formulations.

Development Review of the BACE1 Inhibitor Lanabecestat (AZD3293/LY3314814).

Several ongoing clinical development programs are investigating potential disease-modifying treatments for Alzheimer's disease (AD), including lanabecestat (AZD3293/LY3314814). Lanabecestat is a brain-permeable oral inhibitor of human beta-site amyloid (Aß) precursor protein-cleaving enzyme 1 (BACE1) that reduces Aß production. As a potent BACE1 inhibitor, lanabecestat significantly reduced soluble Aß species and soluble amyloid precursor proteins (sAPPß) in mouse, guinea pig, and dog in a time- and dose-dependent manner. Significant reductions in plasma and cerebrospinal fluid (CSF) Aß1-40 and Aß1-42 were observed in Phase 1 studies of healthy subjects and AD patients treated with lanabecestat. Three lanabecestat trials are ongoing and intended to support registration in Early AD: (1) Phase 2/3 study in patients with mild cognitive impairment (MCI) due to AD and mild AD dementia (AMARANTH, NCT02245737); (2) Delayed-start extension study (AMARANTH-EXTENSION, NCT02972658) for patients who have completed treatment in the AMARANTH Study; and (3) Phase 3 study in mild AD dementia (DAYBREAK-ALZ, NCT02783573). This review will discuss the development of lanabecestat, results from the completed nonclinical and clinical studies, as well as describe the ongoing Phase 3 clinical trials.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Quality requirements for allergen extracts and allergoids for allergen immunotherapy.

All allergen products for allergen immunotherapy currently marketed in the European Union are pharmaceutical preparations derived from allergen-containing source materials like pollens, mites and moulds. Especially this natural origin results in particular demands for the regulatory requirements governing allergen products. Furthermore, the development of regulatory requirements is complicated by the so far missing universal link between certain quality parameters, in particular biological potency, on the one hand and clinical efficacy on the other hand. As a consequence, each allergen product for specific immunotherapy has to be assessed individually for its quality, safety and efficacy. At the same time, biological potency of allergen products is most commonly determined using IgE inhibition assays based on human sera relative to product-specific in house references, ruling out full comparability of products from different manufacturers. This review article aims to summarize the current quality requirements for allergen products including the special requirements implemented for control of chemically modified allergen extracts (allergoids).

Hirschsprung's disease in twins: a systematic review and meta-analysis.

AIM OF THE STUDY: Hirschsprung's disease (HSCR) is known to occur in families. The reported overall incidence of familial cases is 7.6%, with a higher incidence of 15-21% in total colonic aganglionosis and 50% in the rare total intestinal aganglionosis. HSCR is extremely rare in twins. The aim of this study was to systematically analyse the patterns of HSCR in twins published in the literature. METHODS: Electronic databases Pubmed and Medline were screened for relevant articles using the keywords "Hirschsprung's disease", "aganglionosis", "twins", "monozygotic", and "dizygotic". Examining reference lists identified further relevant papers. MAIN RESULTS: Twelve studies with a total of 18 twin pairs were included in this analysis. 67% twins were discordant. HSCR was found in 24 out of 36 twin subjects (67%), of which 83% affected were male. Rectosigmoid type was reported in 71% of patients, long-segment disease in 21, and 8% presented with a total aganglionosis. Three twin pairs had at least one family member affected with HSCR. CONCLUSION: HSCR was found in two-thirds of twin subjects with a male predominance. Rectosigmoid aganglionosis was the most common variant. Disease discordance was identified, where environmental insults were postulated to be predisposing factors to disease expression. Future studies investigating the disease-associated mutations in the already identified HSCR genes should provide insights into the genetic basis of HSCR in twins.

Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

A High Fiber Cookie Made with Resistant Starch Type 4 Reduces Post-Prandial Glucose and Insulin Responses in Healthy Adults.

Distarch phosphate is a resistant starch type 4 (RS4) containing phosphodiester cross-links within and between starch molecules. This study examined the glycemic effects of VERSAFIBE 1490™ resistant starch, a distarch phosphate derived from potato, containing 90% total dietary fiber (TDF, AOAC 991.43 method). In this double-blind, randomized, placebo-controlled, cross-over study, 28 healthy adults consumed a cookie containing 24 g fiber from distarch phosphate (fiber cookie) or a control cookie containing 0.5 g fiber that was matched for fat, protein, and total carbohydrate content. Intravenous blood glucose, intravenous blood insulin, and capillary glucose were measured for two hours after cookie consumption. The fiber cookie reduced the post-prandial blood glucose incremental area under the curve from 0 to 120 minutes (iAUC0-120min) by 44% (p = 0.004) and reduced the maximum glucose concentration (Cmax0-120min) by 8% (p = 0.001) versus the control cookie. Consumption of the fiber cookie resulted in a significant 46% reduction of the post-prandial serum insulin iAUC0-120min (p < 0.001) and a 23% reduction in Cmax0-120min (p = 0.007) versus the control cookie. This study shows that distarch phosphate RS4 can be incorporated into a cookie and significantly reduce post-prandial glucose and insulin responses in healthy adults.

Downregulation of Forkhead box F1 gene expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PURPOSE: High mortality and morbidity in infants born with congenital diaphragmatic hernia (CDH) are attributed to pulmonary hypoplasia and pulmonary hypertension (PH). Forkhead box (Fox) transcription factors are known to be crucial for cell proliferation and homeostasis. FoxF1 is essential for lung morphogenesis, vascular development, and endothelial proliferation. Mutations in FoxF1 and also the Fox family member FoxC2 have been identified in neonates with PH. In human and experimental models of arterial PH, the Fox protein FoxO1 was found to be downregulated. We hypothesized that Fox expression is altered in the lungs of the nitrofen-induced CDH rat model and investigated the expression of FoxF1, FoxC2, and FoxO1. METHODS: Following ethical approval (Rec 913b), time-pregnant Sprague-Dawley rats received nitrofen or vehicle on gestational day (D9). Fetuses were sacrificed on D21, inspected for CDH and divided into CDH (n = 11) and control group (n = 11). Gene expression of FoxF1, FoxC2, and FoxO1 was evaluated with qRT-PCR. Detected alterations of mRNA levels were subsequently assessed on the protein level by performing western blot analysis and laser scanning confocal microscopy. RESULTS: The relative mRNA level of FoxF1 was significantly downregulated in CDH lungs compared to controls (FoxF1 CDH 1.047 ± 0.108, FoxF1 Ctrl 1.419 ± 0.01, p = 0.014). Relative mRNA levels of FoxC2 and FoxO1 were not found to be altered between the experimental groups (FoxC2 CDH 30.74 ± 8.925, FoxC2 Ctrl 27.408 ± 7.487, p = 0.776; FoxO1 CDH 0.011 ± 0.002, FoxO1 Ctrl 0.011 ± 0.001, p = 0.809). On the protein level, western blotting demonstrated a reduced pulmonary protein expression of FoxF1 in CDH lungs. Confocal microscopy showed a markedly diminished expression of FoxF1 in the pulmonary vasculature of CDH lungs compared to controls. CONCLUSION: Our study demonstrates a strikingly reduced expression of FoxF1 in the pulmonary vasculature of nitrofen-induced CDH. Altered FoxF1 gene expression during embryogenesis may participate in vascular maldevelopment resulting in PH in this animal model.

Comparison of MERV 16 and HEPA filters for cab filtration of underground mining equipment.

Significant strides have been made in optimizing the design of filtration and pressurization systems used on the enclosed cabs of mobile mining equipment to reduce respirable dust and provide the best air quality to the equipment operators. Considering all of the advances made in this area, one aspect that still needed to be evaluated was a comparison of the efficiencies of the different filters used in these systems. As high-efficiency particulate arrestance (HEPA) filters provide the highest filtering efficiency, the general assumption would be that they would also provide the greatest level of protection to workers. Researchers for the U.S. National Institute for Occupational Safety and Health (NIOSH) speculated, based upon a previous laboratory study, that filters with minimum efficiency reporting value, or MERV rating, of 16 may be a more appropriate choice than HEPA filters in most cases for the mining industry. A study was therefore performed comparing HEPA and MERV 16 filters on two kinds of underground limestone mining equipment, a roof bolter and a face drill, to evaluate this theory. Testing showed that, at the 95-percent confidence level, there was no statistical difference between the efficiencies of the two types of filters on the two kinds of mining equipment. As the MERV 16 filters were less restrictive, provided greater airflow and cab pressurization, cost less and required less-frequent replacement than the HEPA filters, the MERV 16 filters were concluded to be the optimal choice for both the roof bolter and the face drill in this comparative-analysis case study. Another key finding of this study is the substantial improvement in the effectiveness of filtration and pressurization systems when using a final filter design.

Long-term results of transanal pull-through for Hirschsprung's disease: a meta-analysis.

INTRODUCTION: Several operative techniques have been developed for the treatment of Hirschsprung's disease (HD) in the past decades. Since one-stage transanal pull-through (TAPT) was first performed in 1998, multiple studies have shown favourable short-and midterm results compared to other techniques with shorter operation length, shorter hospital stay and lower complication rates. The aim of this meta-analysis was to determine the longterm results following TAPT for HD. METHODS: A systematic literature search for relevant articles was performed in four databases using the following terms "Hirschsprung/Hirschsprung's disease", "aganglionosis", "transanal", "pullthrough/pull-through", "longterm/long-term" "results", "follow-up" and "outcome". A meta-analysis was conducted for relevant articles for one-stage transanal pull-through for HD with a minimal follow-up of median 36 months regarding constipation, incontinence/soiling, enterocolitis and secondary operations. Odds ratio (OR) with 95 % confidence intervals (CI) were calculated. RESULTS: Six studies with 316 patients matched the set criteria and were included in this analysis. Overall 45 (14.2 %) patients had disturbances of bowel function (OR 0.05, 95 % CI 0.03-0.07, p < 0.00001). Of these, 24 (53.3 %) patients experienced constipation, 8 (17.8 %) incontinence/soiling and 13 (28.9 %) enterocolitis. 10 (3.2 %) patients developed complications requiring secondary surgery. Most patients had a daily defecation frequency of 1-3 bowel movements 3 years postoperatively, resembling the stooling patterns of healthy controls. CONCLUSION: Nearly 15 % of all patients operated with TAPT for HD continue to experience persistent bowel symptoms with constipation as the main problem. Further studies on the long-term outcome of children operated with this technique for HD are necessary to evaluate stooling patterns, urinary and sexual function as well as general quality of life during adolescence and adulthood.

Laparoscopic-assisted pull-through operation for Hirschsprung's disease: a systematic review and meta-analysis.

PURPOSE: In the last two decades, laparoscopic-assisted pull-through (LAPT) has gained much popularity in the treatment of Hirschsprung's disease. The aim of this meta-analysis was to determine the long-term outcome of patients treated laparoscopically. METHODS: A systematic literature-based search for relevant cohorts was performed using the terms "Hirschsprung's disease and Laparoscopy", "Laparoscopic-assisted pull-through outcome", "Laparoscopic-assisted Soave pull-through" "Laparoscopic-assisted Swenson pull-through" and Laparoscopic-assisted Duhamel pull-through. The relevant cohorts of laparoscopic operated HD were systematically searched for outcome regarding continence, constipation, secondary surgery related to the laparoscopic approach and enterocolitis. Pooled incidence rates and odds ratios (ORs) with 95 % confidence intervals (CI) were calculated using standardized statistical methodology. RESULTS: Sixteen studies met defined inclusion criteria, reporting a total of 820 patients. All studies were retrospective case series, with variability in outcome assessment quality and length of follow-up. The median cohort size consisted of 28 patients (range 15-218). In the long-term follow-up, 97 patients (11.14 %) experienced constipation (OR 0.06, 95 % CI 0.05-0.08, p < 0.00001), 53 (6.46 %) incontinence/soiling (OR 0.01 95 % CI 0.01-0.01, p < 0.00001), 75 (9.14 %) recurrent enterocolitis (OR 0.02 95 % CI 0.01-0.02, p < 0.00001) and 69 (8.4 %) developed complications requiring secondary surgery (OR 0.01 95 % CI 0.01-0.02, p < 0.00001). Overall events in long-term follow-up occurred in 225 (27.5 %) patients (OR 0.24 95 % CI 0.20-0.30, p < 0.00001). CONCLUSIONS: This meta-analysis shows that nearly one-third of the patients continue to have long-term bowel problems, such as constipation, soiling and recurrent enterocolitis following LAPT. Many patients treated by LAPT require secondary surgery. Large randomized studies with long-term follow-up are necessary to determine the difference in outcome between LAPT and completely transanal pull-through operation.