Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

The features of the reparative regeneration of an oral mucosa wound created under the exposure of a laser at a wavelength of 445 nm (a pilot study).

In clinical practice, an innovative laser technology that provides contactless preparation of soft tissues with a wavelength of 445 nm has been introduced. This study aimed to investigate the morphological changes in the oral mucosa when exposed to laser radiation at a wavelength of 445 nm in the ablation mode.An experimental study was conducted to analyze the dynamics of reparative regeneration in the wound caused by that particular type of radiation, utilizing the procedure of lower lip frenuloplasty as an illustration. 48 sexually mature male laboratory rats were chosen as the research object. The procedure of preparing the oral vestibule was executed by employing a contactless laser beam with a wavelength of 445 nm and a power of 0.7 W in continuous mode (CW) and an uninitiated fiber.Histological examination showed that 25 min after the surgery, there were large areas of coagulation necrosis in the oral mucosa in the area affected by the blue laser. In 48 h, the area of necrosis decreased both in size and depth. By the 7th day after the surgery, the necrotic masses had grown into the connective tissue, while marginal regeneration of the epithelium was noted. By the 14th day, the wound surface was completely epithelialized, represented by fibrous scar tissue. Clinically, around the mandibular incisors, there was a wide area of attached keratinized gingiva.The findings of histological examination indicate a necrosis of coagulation type in the region of tissue ablation and also show the absence of phase II of the inflammatory response (the stage of exudation), which expedites the process of epithelialization of the oral mucosa wound.

Monolacunary Wells-Dawson Polyoxometalate as a Novel Contrast Agent for Computed Tomography: A Comprehensive Study on In Vivo Toxicity and Biodistribution.

Polyoxotungstate nanoclusters have recently emerged as promising contrast agents for computed tomography (CT). In order to evaluate their clinical potential, in this study, we evaluated the in vitro CT imaging properties, potential toxic effects in vivo, and tissue distribution of monolacunary Wells-Dawson polyoxometalate, α2-K10P2W17O61.20H2O (mono-WD POM). Mono-WD POM showed superior X-ray attenuation compared to other tungsten-containing nanoclusters (its parent WD-POM and Keggin POM) and the standard iodine-based contrast agent (iohexol). The calculated X-ray attenuation linear slope for mono-WD POM was significantly higher compared to parent WD-POM, Keggin POM, and iohexol (5.97 ± 0.14 vs. 4.84 ± 0.05, 4.55 ± 0.16, and 4.30 ± 0.09, respectively). Acute oral (maximum-administered dose (MAD) = 960 mg/kg) and intravenous administration (1/10, 1/5, and 1/3 MAD) of mono-WD POM did not induce unexpected changes in rats' general habits or mortality. Results of blood gas analysis, CO-oximetry status, and the levels of electrolytes, glucose, lactate, creatinine, and BUN demonstrated a dose-dependent tendency 14 days after intravenous administration of mono-WD POM. The most significant differences compared to the control were observed for 1/3 MAD, being approximately seventy times higher than the typically used dose (0.015 mmol W/kg) of tungsten-based contrast agents. The highest tungsten deposition was found in the kidney (1/3 MAD-0.67 ± 0.12; 1/5 MAD-0.59 ± 0.07; 1/10 MAD-0.54 ± 0.05), which corresponded to detected morphological irregularities, electrolyte imbalance, and increased BUN levels.

Diurnal changes in bacterial settlement on the Peyer's patch and surrounding mucosa in the rat ileum and its effect against the intestinal immune system.

Our previous study revealed the diurnal change in the indigenous bacteria settling on the terminal region of the rat ileum. In the present study, we investigated the diurnal change in indigenous bacteria on the most distal ileal Peyer's patch (PP) and surrounding ileal mucosa and explored how stimulation from indigenous bacteria for a day affects the intestinal immune system at the beginning of the light phase. Histological measurement revealed that bacteria adjacent to the follicle-associated epithelium of PP and to the villous epithelium of the surrounding ileal mucosa are more abundant at zeitgeber time (ZT)0 and ZT18 than at ZT12. On the other hand, tissue-section 16S rRNA amplicon sequencing revealed no significant difference between ZT0 and ZT12 in the bacterial composition on the ileal tissue including the PP. One-day treatment with an antibiotic (Abx) successfully impaired the settlement of bacteria around the ileal PP. In transcriptome analysis, 1-day Abx treatment led to the downregulation of several chemokines in both PP and ordinary ileal mucosa at ZT0. Histological analysis of the 1-day Abx group revealed decreases in both CD68+ macrophages in PP and naphthol AS-D chloroacetate esterase stain-positive mast cells in the ileal villi. Together, these findings suggest that the colonies of indigenous bacteria on the distal ileal PP and surrounding mucosa expand during the dark phase, which might lead to the expression of genes to regulate the intestinal immune system and contribute to the homeostasis of at least macrophages in PP and mast cells in the ileal mucosa.

Multimethod combination for age estimation of Sarcophaga peregrina (Diptera: Sarcophagidae) with implications for estimation of the postmortem interval.

Sarcophaga peregrina (Robineau-Desvoidy, 1830) (Diptera: Sarcophagidae) is a forensically important flesh fly with potential value for estimating the minimum postmortem interval (PMImin). Here, the developmental patterns of S. peregrina were investigated at 5 constant temperatures (15-35 °C). Morphological changes at different developmental stages of the pupa were observed at 4 constant temperatures (15-30 °C) by removing the puparium and staining the pupa with hematoxylin and eosin. Furthermore, differentially expressed genes (DEGs) were analyzed at 25 °C in the intrapuparial period to estimate the age of S. peregrina during the intrapuparial stage. S. peregrina completed development from larviposition to adult eclosion at 15 °C, 20 °C, 25 °C, and 30 °C; the developmental durations were 1090.3 ± 30.6 h, 566.6 ± 21.9 h, 404.6 ± 13.01 h, and 280.3 ± 4.5 h, respectively, while the development could not be completed at 35 °C. The intrapuparial period of S. peregrina was divided into 12 sub-stages on the basis of the overall external morphological changes; 6 sub-stages on the basis of individual morphological structures such as the compound eyes, antennae, thorax, legs, wings, and abdomen; and 10 sub-stages on the basis of internal morphological changes detected using histological analysis. The period of each sub-stage or structure that appeared was determined. Moreover, we found that 6 genes (NDUFS2, CPAMD8, NDUFV2, Hsp27, Hsp23, and TPP) with differential expression can be used for the precise age estimation of S. peregrina during the intrapuparial period. This study provided basic developmental data for the use of S. peregrina in PMImin estimation, and we successfully estimated PMImin in a real forensic case by using a multimethod combination.

Prognostic and biological function value of OSBPL3 in colorectal cancer analyzed by multi-omic data analysis.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies in the world. This study proposes to reveal prognostic biomarkers for the prognosis and treatment of CRC patients. METHODS: Differential analysis of OSBPL3 was performed in pan-cancer, and the correlation between clinical stage and OSBPL3 was analyzed. Multiple omics analysis was used to compare the relationship between survival of patients and copy number variation, single nucleotide variant, and methylation status. Survival differences between high and low OSBPL3 expression groups were analyzed. Differentially expressed genes (DEGs) between high and low OSBPL3 expression groups were obtained, and functional enrichment analysis was implemented. Correlations between immune cells and OSBPL3 was analyzed. Drug sensitivity between the two OSBPL3 expression groups was compared. Moreover, the expression of OSBPL3 was verified by immunohistochemistry and real-time quantitative PCR. RESULTS: OSBPL3 was differentially expressed in 13 tumors and had some correlations with T and N stages. OSBPL3 expression was regulated by methylation and higher OSBPL3 expression was associated with poorer prognosis in CRC. 128 DEGs were obtained and they were mainly involved in signaling receptor activator activity, aspartate and glutamate metabolism. T cell gamma delta and T cell follicular helper were significantly different in the high and low OSBPL3 expression groups. Moreover, OSBPL3 showed negative correlations with multiple drugs. OSBPL3 was significantly upregulated in CRC samples compared to normal samples. CONCLUSIONS: A comprehensive analysis demonstrated that OSBPL3 had potential prognostic value, and guiding significance for CRC chemotherapeutic.

Heparin coated decellularized xenogeneic small diameter vascular conduit for vascular repair with early luminal reendothelialization.

Small diameter vascular graft is a clinical need in cardiovascular disease (CAD) and peripheral atherosclerotic diseases (PAD). Autologous graft has limitations in availability and harvesting surgery. To make luminal surface modification with heparin coating in xenogeneic small diameter vascular graft. We constructed a conduit from decellularized human saphenous vein (HSV) matrices in small diameter vascular graft (< 0.8 mm diameter). Luminal surface modification was done with heparin coating for transplantation in the rat femoral artery. Biocompatibility of conduit was checked in Chorioallantoic Membrane (CAM) assay and in vivo. The blood flow rate in conduit grafts was measured, and immuno-histological analysis was performed. CAM assay and in vivo biocompatibility test showed cellular recruitment in the HSV scaffold. Heparin binding was achieved on the luminal surface. After three months of transplantation surgery neo-intimal layer was formed in the graft. The graft was patent for two weeks after surgery. There were no statistically differences between blood flow rate in graft (at proximal end 0.5 ± 0.01 m/s and at distal end 0.4 ± 0.01 m/s (n = 6)) and native artery (0.6 ± 0.1 m/second, (n = 3)). Biomarkers of endothelial cells, medial smooth muscle cells, and angiogenesis were observed in the transplanted graft. Our study demonstrates that xenogeneic decellularized vascular grafts with surface modification with heparin coating could be useful for the replacement of small diameter vessels.

Histological analysis for pulp mineralisation after severe intrusive luxation of immature molars in rats.

BACKGROUND/AIM: Pulp mineralisation is a survival process that may occur in the pulp of immature teeth following trauma. However, the mechanism of this process remains unclear. The aim of this study was to evaluate the histological manifestations of pulp mineralisation after intrusion in immature molars of rats. MATERIALS AND METHODS: Three-week-old male Sprague-Dawley rats were subjected to intrusive luxation of the right maxillary second molar by an impact force from a striking instrument through a metal force transfer rod. The left maxillary second molar of each rat was used as a control. The control and injured maxillae were collected at 3, 7, 10, 14, and 30 days after trauma (n = 15 per time group) and evaluated using haematoxylin and eosin staining and immunohistochemistry. Independent two-tailed Student's t-test was used for statistical comparison of the immunoreactive area. RESULTS: Pulp atrophy and mineralisation were observed in 30%-40% of the animals, and no pulp necrosis occurred. Ten days after trauma, pulp mineralisation, with osteoid tissue rather than reparative dentin, formed around the newly vascularised areas in the coronal pulp. CD90-immunoreactive cells were observed in the sub-odontoblastic multicellular layer in control molars, whereas the number of these cells was decreased in the traumatised teeth. CD105 localised in cells around the pulp osteoid tissue of the traumatised teeth, whereas in control teeth, it was only expressed in the vascular endothelial cells of capillaries in the odontoblastic or sub-odontoblastic layers. In specimens with pulp atrophy at 3-10 days after trauma, hypoxia inducible factor expression and CD11b-immunoreactive inflammatory cells increased. CONCLUSIONS: Following intrusive luxation of immature teeth without crown fractures in rats, no pulp necrosis occurred. Instead, pulp atrophy and osteogenesis around neovascularisation with activated CD105-immunoreactive cells were observed in the coronal pulp microenvironment characterised by hypoxia and inflammation.

Histological study of diurnal changes in bacterial settlement in the rat alimentary tract.

The composition of fecal bacteria is reported to change throughout the day, whereas the circadian rhythmicity of indigenous bacteria that settle on the epithelium is mostly unknown. The present study aimed to clarify the diurnal changes in the settlement of indigenous bacteria in the rat alimentary tract using histological analysis. The settlement of indigenous bacteria on the mucosal epithelium throughout the day and the diurnal changes in settlement levels were observed in the esophagus, the nonglandular area of the stomach, and the ileum. The peak of zeitgeber time (ZT) in the settlement level differed by segment: ZT 12 in the esophagus, ZT 6 in the nonglandular area of the stomach, and ZT 0 in the ileum. Moreover, 16S rRNA amplicon sequencing using tissue sections revealed that the compositions of the indigenous bacteria in the ileum differed among ZT. In the intervillous spaces of the ileum, the formation level of the mucus layer, one of the most fundamental host defenses against bacteria, was lowest at ZT 0. Bacteria were preferentially adjacent to the villous epithelium in the area without coverage by the mucus layer at ZT 0. These findings collectively suggest that the settlement level and possibly the composition of the indigenous bacteria changed diurnally in various segments of the alimentary tract, and the formation of the mucus layer might be the most likely to lead to such diurnal changes in indigenous bacteria, at least in the ileum.

Treatment of protoscoleces with gamma radiation: Potential immunoprotective effect against experimental murine echinococcosis.

Cystic echinococcosis (CE) is one of the most important zoonotic diseases with a worldwide distribution. It is caused by the larval stage of the dog tapeworm "Echinococcus granulosussensu lato" and constitutes a major economic and public health problem in several countries. Protoscoleces are one component of this larval stage that can interact with both definitive and intermediate hosts. The aim of the present study was to investigate the potential role of using a radio-attenuated form of these protoscoleces for immunoprophylaxis against experimental murine echinococcosis. However, mice were immunized twice at 15-day intervals with gamma (γ) irradiated protoscoleces at doses of 0.4, 0.8, 1.2 and 1.4 kGy then challenged with the intact parasites. Macroscopic and histological analyses with cytokine measurements were performed in order to estimate the number and diameter of cysts, microscopic changes and cytokine profile. An improvement in protection against the challenge dose was observed with increasing dose, giving percentages of 47.7, 49, 55.23 and 70.6%, for the 0.4, 0.8, 1.2 and 1.4 kGy-groups respectively. These data suggest that immunization with radio-attenuated protoscoleces may induce satisfactory protective immunity by reducing successfully the formation of cysts, caused by challenge infection.

Evidence of Anti-Inflammatory Effect of Transcranial Direct Current Stimulation in a CFA-Induced Chronic Inflammatory Pain Model in Wistar Rats.

INTRODUCTION: Given that chronic inflammatory pain is highly prevalent worldwide, it is important to study new techniques to treat or relieve this type of pain. The present study evaluated the effect of transcranial direct current stimulation (tDCS) in rats submitted to a chronic inflammatory model by nociceptive response, biomarker levels (brain-derived neurotrophic factor [BDNF] and interleukin [IL]-6 and IL-10), and by histological parameters. METHODS: Sixty-day-old male Wistar rats were used in this study and randomized by weight into 6 major groups: total control, control + sham-tDCS, control + active tDCS, total CFA, CFA + sham-tDCS, and CFA + active tDCS. After inflammatory pain was established, the animals were submitted to the treatment protocol for 8 consecutive days, according to the experimental group. The nociceptive tests (von Frey and hot plate) were assessed, and euthanasia by decapitation occurred at day 8 after the end of tDCS treatment, and the blood serum and central nervous structures were collected for BDNF and IL measurements. All experiments and procedures were approved by the Institutional Committee for Animal Care and Use (UFPel #4538). RESULTS: The tDCS treatment showed a complete reversal of the mechanical allodynia induced by the pain model 24 h and 8 days after the last tDCS session, and there was partial reversal of the thermal hyperalgesia at all time points. Serum BDNF levels were decreased in CFA + sham-tDCS and CFA + tDCS groups compared to the control + tDCS group. The control group submitted to tDCS exhibited an increase in serum IL-6 levels in relation to the other groups. In addition, there was a significant decrease in IL-10 striatum levels in control + tDCS, CFA, and CFA + sham-tDCS groups in relation to the control group, with a partial tDCS effect on the CFA pain model. Local histology demonstrated tDCS effects in decreasing lymphocytic infiltration and neovascularization and tissue regeneration in animals exposed to CFA. CONCLUSION: tDCS was able to reverse the mechanical allodynia and decrease thermal hyperalgesia and local inflammation in a chronic inflammatory pain model, with a modest effect on striatum IL-10 levels. As such, we suggest that analgesic tDCS mechanisms may be related to tissue repair by modulating the local inflammatory process.

What happens to brain outside the thermal ablation zones? An assessment of needle-based therapeutic ultrasound in survival swine.

BACKGROUND: In stereotactic radiosurgery, isodose lines must be considered to determine how surrounding tissue is affected. In thermal ablative therapy, such as laser interstitial thermal therapy (LITT), transcranial MR-guided focused ultrasound (tcMRgFUS), and needle-based therapeutic ultrasound (NBTU), how the surrounding area is affected has not been well studied. OBJECTIVE: We aimed to quantify the transition zone surrounding the ablation core created by magnetic resonance-guided robotically-assisted (MRgRA) delivery of NBTU using multi-slice volumetric 2-D magnetic resonance thermal imaging (MRTI) and subsequent characterization of the resultant tissue damage using histopathologic analysis. METHODS: Four swine underwent MRgRA NBTU using varying duration and wattage for treatment delivery. Serial MRI images were obtained, and the most representative were overlaid with isodose lines and compared to brain tissue acquired postmortem which underwent histopathologic analysis. These results were also compared to predicted volumes using a finite element analysis model. Contralateral brain tissue was used for control data. RESULTS: Intraoperative MRTI thermal isodose contours were characterized and comprehensively mapped to post-operative MRI images and qualitatively compared with histological tissue sections postmortem. NBTU 360° ablations induced smaller lesion volumes (33.19 mm3; 120 s, 3 W; 30.05 mm3, 180 s, 4 W) versus 180° ablations (77.20 mm3, 120 s, 3 W; 109.29 mm3; 180 s; 4 W). MRTI/MRI overlay demonstrated the lesion within the proximal isodose lines. The ablation-zone was characterized by dense macrophage infiltration and glial/neuronal loss as demonstrated by glial fibrillary acidic protein (GFAP) and neurofilament (NF) absence and avid CD163 staining. The transition-zone between lesion and normal brain demonstrated decreased macrophage infiltration and measured ∼345 microns (n - 3). We did not detect overt hemorrhages or signs of edema in the adjacent spared tissue. CONCLUSION: We successfully performed MRgRA NBTU ablation in swine and demonstrated minimal histologic changes extended past the ablation-zone. The lesion was characterized by macrophage infiltration and glial/neuronal loss which decreased through the transition-zone.

Soft tissue response to different abutment materials: A controlled and randomized human study using an experimental model.

OBJECTIVES: Aim of this study was to compare the soft tissue response to implant abutments made of titanium, zirconia, zirconia veneered with feldspar ceramics and PEEK by various clinical, histological, microbiological, and molecular biological markers in an experimental model. MATERIALS AND METHODS: A total of 40 experimental one-piece healing abutments of four different materials were mounted on bone level implants in 20 volunteering patients (split-mouth design). After a three-month period of open healing, clinical parameters at the abutments were assessed and adjacent mucosa was sampled for inflammatory cytokine mRNA concentrations and histological analysis by a novel method. In addition, PISF samples were obtained for the analysis of periodonto-pathogenic bacteria counts and active MMP-8 levels. Marginal bone level change was measured by intra oral radiographs. RESULTS: Abutments of the different materials did not exhibit significant differences regarding clinical parameters, pathogenic bacteria counts or pro-inflammatory cytokine concentrations. Likewise, no significant differences were detected regarding soft tissue morphology or bone level change. Compared to titanium abutments, significantly less mononuclear inflammatory cells were detected in the mucosa at abutments made of zirconia veneered with feldspar ceramics. CONCLUSIONS: All examined abutment materials exhibited a similar soft tissue response compared to titanium and histological data did not reveal early signs of elevated inflammation caused by PEEK- and feldspar-veneered zirconia abutments. Due to the short observation period and the small sample size, a final conclusion on the long-term suitability of those abutment materials cannot be drawn. However, based on the presented data, we consider further studies on that subject as appropriate.

Comparison of lipid metabolism between broodstock and hybrid offspring in the hepatopancreas of juvenile shrimp (Macrobrachium nipponense): Response to chronic ammonia stress.

Ammonia nitrogen is a major pollutant that causes great physiological harm to crustaceans in culture. In this study, we conducted a 28 day chronic ammonia nitrogen stress experiment with broodstock populations (Dianshan, DS) and hybrid offspring populations (DS ♀ × CD (Changjiang ♂ × Dongting ♀), SCD) exposed to 0, 1 and 10 mg/L of ammonia concentrations. A 28 day feeding trial and chronic ammonia nitrogen stress were used to investigate the effects on the growth performance, histological structure and lipid metabolism of juvenile shrimp, Macrobrachium nipponense. Our results indicated that survival rates in the SCD groups were significantly higher than those in the DS groups, whereas weight and length gain rates were not significantly different between the groups (p > 0.05). Histological structure results showed that the number of vacuoles in the DS group was significantly higher than that in the SCD group and hepatopancreas cell structures were disrupted in the ammonia treatment groups. The results of oil red staining showed that the number of lipid droplets increased significantly with the increase in ammonia concentration. As the ammonia concentration increased, fatty acid contents, lipid enzyme activities and lipid metabolism-related gene expression all tended to rise. In conclusion, ammonia nitrogen exposure caused damage to the hepatopancreas structure of juvenile shrimp and disturbed the lipid metabolism of the hepatopancreas. In addition, the SCD population had stronger stress resistance than the DS population when subjected to the same concentration of ammonia nitrogen stress.

Assessment of antiandrogenic and antispermatogenic activity of Hedera nepalensis in adult male rats.

The use of medicinal plants for fertility regulation has been prevalent worldwide for many centuries. They possess natural substances having antiandrogenic properties and can be used as source of contraception. In the current study, methanolic leaf extract of Hedera nepalensis was evaluated for antiandrogenic and antispermatogenic activity in adult male rats through various reproductive parameters. Experimental findings showed significantly increased oxidative stress with reduced antioxidant activity at highest dose regimens in both in vitro and in vivo studies. Increased ROS generation and lipid peroxidation lead to DNA damage in rat sperm. In vivo determination of sperm parameters exhibited notable reduction in sperm motility, viability and DSP in dose-treated animals. Histopathological observations revealed reduced epithelial height and wider lumen having less number of spermatozoa in high-dose-treated groups. Additionally, a marked decline noted in Testosterone concentration in all extract treated groups, while plasma LH and FSH levels only in high-dose-treated groups were noted. The findings of the current study conclude that methanolic leaf extract of H. nepalensis has the potential to disturb male fertility by generating oxidative stress and hormonal imbalance leading to histological alterations and sperm DNA damage.

Evaluating the efficacy of anti-fungal blue light therapies via analyzing tissue section images.

Anti-fungal blue light (ABL) therapies have been studied and applied in treating various diseases caused by fungal infection. The existing work has been mainly devoted to study the effect of various light dosages on the fungal viability and on the induced cytotoxic reactive oxygen species (ROS) in the pathogens. While in vivo experimental studies have also been reported, there is still no work targeted on quantifying the effect of light on prohibiting the pathogens from invading into the deeper sites in the skin of their host. This can be attributed to the lack of methods to analyze the tissue section images, which are the main means of examining infected tissues. This work has been devoted to solve such problems, so as to improve dosimetric analyses of ABL therapies on treating fungal infections. Specifically, the invasion depth of the fungi and their ratios to the tissue in four bins at different depths inside the skin were extracted from the tissue section images. The significance of the treatment with different dosages on inhibiting the fungi was also tested by each of these depth-related metrics. The ABL experiments using 415-nm-wavelength LED light were performed on BALB/c mice, whose skin was infected by Candida albicans (C. albicans). The proposed methods were applied to the tissue sections of the experimental animals. The results clearly verified that the fluence up to 180J/cm2 can significantly prohibit the fungal infection into the skin in terms of almost all the newly proposed metrics.

Reproductive biology of the bullseye puffer Sphoeroides annulatus: Gonadosomatic index and its suitability for estimating length at maturity.

This study aimed to describe updated information on the basic reproductive biology of Sphoeroides annulatus females and potential use of the gonadosomatic index I G as input data to assess length at 50% maturity ( L 50 ) and identify the breeding season. From April 2014 to December 2015, 767 females were analysed along the north-west coast of the Mexican Pacific Ocean. Both immature and mature females were represented in the total length ( L T ), which ranged from 81 to 440 mm, and the smallest mature female was 92 mm (new record for the species). Gonadal maturity phases were assigned through histological analysis. A I G threshold value I cut was established using the lower whisker value for maturity phase spawning capable (2.06%). The monthly variation of I G supported the annual spawning seasonality, and the presence of reproductively active mature females I G ≥ I cut coincided with the observed periods with histological data and the peaks for both seasons 2014 and 2015. Also the presence of reproductively inactive I G < I cut mature females confirmed the nonreproductive season observed from January to April 2015. Estimates of L 50 were based on the cumulative proportion of reproductively active mature females: both datasets showed a 'bumpy trajectory'. Considering the functional form of data and models, a second objective was to verify if each dataset presented a simple or double sigmoid trajectory and select the most suitable model through the Akaike information criterion. For both datasets, the best fitted model was the double sigmoidal M DS , while the single sigmoidal M SS had low performance and did not have statistical support. The M DS provided a L 50 value ca. 254 mm L T for both data sets. A trade-off between the model's correct fit and a reliable L 50 value was found, but this fact is not always considered when calculating the maturity, resulting in biased parameters. Correct data fitting may seem trivial, but the consequences for the conservation of immature individuals could be dramatic, affecting the population recruitment. In conclusion, the histologically calibrated I cut was suitable for classifying females of S. annulatus into reproductively inactive or active types, with an agreement percentage of 99%. The I cut  = 2.06% can be useful for subsequent studies for this species.

Analysis of histology and long noncoding RNAs involved in the rabbit hair follicle density using RNA sequencing.

BACKGROUND: Hair follicle density influences wool fibre production, which is one of the most important traits of the Wan Strain Angora rabbit. However, molecular mechanisms regulating hair follicle density have remained elusive. RESULTS: In this study, hair follicle density at different body sites of Wan Strain Angora rabbits with high and low wool production (HWP and LWP) was investigated by histological analysis. Haematoxylin-eosin staining showed a higher hair follicle density in the skin of the HWP rabbits. The long noncoding RNA (lncRNA) profile was investigated by RNA sequencing, and 50 and 38 differentially expressed (DE) lncRNAs and genes, respectively, were screened between the HWP and LWP groups. A gene ontology analysis revealed that phospholipid, lipid metabolic, apoptotic, lipid biosynthetic, and lipid and fatty acid transport processes were significantly enriched. Potential functional lncRNAs that regulate lipid metabolism, amino acid synthesis, as well as the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) and hedgehog signalling pathways, were identified. Consequently, five lncRNAs (LNC_002171, LNC_000797, LNC_005567, LNC_013595, and LNC_020367) were considered to be potential regulators of hair follicle density and development. Three DE lncRNAs and genes were validated by quantitative real-time polymerase chain reaction (q-PCR). CONCLUSIONS: LncRNA profiles provide information on lncRNA expression to improve the understanding of molecular mechanisms involved in the regulation of hair follicle density.

I've got you under my skin: inflammatory response to elephant seal's lice.

Seals (Phocidae) undergo an annual cycle of moulting that implies hair regeneration, and in the case of southern elephant seals, it also involves the superficial strata of the epidermis. Therefore, surviving the moulting period is crucial for their obligate and permanent ectoparasites. Throughout evolutionary time, sucking lice (Echinophtiriidae) have developed morphological, behavioural and ecological adaptations to cope with the amphibious lifestyle of their hosts. Lepidophthirus macrorhini, the Southern elephant seal louse species, faces the additional challenge of surviving attached to the host during the moulting period. Since lice live on the skin, L. macrorhini has developed a unique survival strategy by piercing the skin of their host, thus keeping them protected from moulting. During fieldwork in Patagonia and Antarctica, skin samples with lice within were collected for histological analysis to assess whether these parasites caused damage to the host. Lice generate an inflammatory process in the host's dermis, and these lesions could alter the normal chemical and mechanical protective properties of the skin facilitating secondary infections. Further studies that analyse the potential pathogens in those skin lesions are necessary to properly assess the real impact of ectoparasites on their host health.

Novel method to obtain human non-separated histological samples for the assessment of peri-implant soft tissue response: A feasibility study.

OBJECTIVES: To introduce a standardized and less invasive clinical model that provides histological information on the abutment-mucosa interface in humans. MATERIALS AND METHODS: New experimental healing abutments were left in an open healing position on bone-level implants in the interforaminal region of the mandibles in six edentulous patients. The one-piece abutments were hollow cylinder-shaped with two lateral openings that allow for ingrowth of the peri-implant mucosa into the central abutment cavity. After three months of healing, abutments and ingrown mucosa were sampled and processed for histological analysis in a non-separated resin-embedding technique. To test the validity of the new model, the ingrown tissue was compared to the peri-implant mucosa around the same samples. RESULTS: None of the experimental abutments exhibited signs of failure, and all samples showed mucosal ingrowth to the inner-abutment cavity. Comparison of ingrown tissue and peri-implant mucosa revealed no significant differences regarding the traits: tissue morphology, quality of collagen fibers, and adherence to the abutment. Ingrown mucosa exhibited a tendency for higher leukocyte infiltration. CONCLUSIONS: The presented model is a promising approach to reduce invasiveness during the sampling process for human non-separated abutment biopsies.