Fine Structure of Maxillary Palps in Adults of Hermetia illucens (Diptera: Stratiomyidae).

A relevant species in waste management but also in forensic, medical, and veterinary sciences is the black soldier fly, Hermetia illucens (Linnaeus; Diptera: Stratiomyidae). An ultrastructural study by scanning electron microscopy (SEM) was conducted for the first time on maxillary palps of both sexes, describing in detail the morphology and distribution of sensilla and microtrichia. The maxillary palps, composed of two segments, show sexual dimorphism in length and shape. In both sexes, the first segment is covered only by microtrichia, but the second one is divided into two parts: the proximal one, covered only by microtrichia, and the distal one containing both microtrichia and sensory structures. These structures include two types of sensory pits and one of chaetic sensilla. Due to sexual dimorphism in palp size, females have a higher number of sensory pits. The sexual dimorphism of palps and the presence and role of sensilla in H. illucens was discussed in comparison to other species of the family Stratiomyidae and of other Diptera. This study may represent a base for further investigations on mouthpart structures of this species, involved in key physiological activities, such as feeding, mating and oviposition.

Expression analysis of Toll like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure.

Six male Tharparkar cattle of 2-3 years old were selected for the study. After 15 days acclimation at thermo neutral zone (TNZ) in psychrometric chamber, animals were exposed at 42°C for 6h up to 23 days followed by 12 days of recovery period. Blood samples were collected during control period at TNZ (day 1, 5 and 12), after heat stress exposure (day 1-10, Short Term Heat Stress Acclimation - STHSA; day 15-23, Long Term Heat Stress Acclimation - LTHSA) and recovery period (day 7 and 12) and peripheral blood mononuclear cells (PBMCs) were isolated for RNA and protein extraction. Serum cortisol concentration was assessed by RIA. The mRNA and protein expression in PBMCs were determined by qPCR and western blot respectively. Samples at TNZ were taken as control. Serum cortisol concentration was increased (P<0.05) during STHSA and gradually declined during LTHSA. Toll like receptor 2 (TLR 2) expression was up regulated (P<0.05) during STHSA and declined to basal level during LTHSA and recovery phase. However, toll like receptor 4 (TLR 4) expression was up regulated (P<0.05) during STHSA and LTHSA while declined in recovery phase. Interleukin 2 (IL2) and interleukin 6 (IL 6) were up regulated (P<0.05) during STHSA and reduced to basal level during LTHSA. PBMCs culture study was conducted to study transcriptional abundance of TLR2/4 and IL2/6 at different temperature-time combinations. The present findings indicate that TLR 2/4 and IL 2/6 could possibly play a vital role in thermo tolerance in Tharparkar cattle during short term and long term heat stress exposure.

Expression dynamics of HSP70 during chronic heat stress in Tharparkar cattle.

Six male Tharparkar cattle aged 2-3 years were selected for the study. The animals were acclimatized in the psychrometric chamber at thermoneutral zone (TNZ) for 15 days and then exposed to 42 °C temperature up to 23 days followed by 12 days of recovery period. Physiological responses were estimated, and peripheral blood mononuclear cells (PBMCs) were isolated at TNZ on day 1, day 5, and day 12; after 6 h of heat stress exposure on day 16 to day 20, day 25, day 30, day 32, day 34, day 36, and day 38; and a recovery period on day 45 and day 50. The PBMCs were cultured to study the effect of thermal challenge on HSP70 messenger RNA (mRNA) expression pattern at different temperature-time combinations. The mRNA and protein expression of HSP70 in PBMCs along with serum extracellular HSP70 (eHSP70) was increased (P < 0.05) and showed two peaks on day 17 and day 32 (2nd and 17th days of thermal challenge, respectively). The HSP70 mRNA expression was increased (P < 0.05) in a temperature- and time-dependent manner in heat stress challenge treatment as compared to control in cultured PBMCs. HSP70 expression was found to be higher (P < 0.05) after 10 days of heat exposure (corresponds to chronic heat stress) as compared to the first 5 days of heat stress (corresponds to short-term heat stress) and control period at TNZ. The present findings indicate that HSP70 is possibly involved in heat stress adaptive response in Tharparkar cattle and the biphasic expression pattern may be providing a second window of protection during chronic heat stress.

Expression and localization of fibroblast growth factor (FGF) family in buffalo ovarian follicle during different stages of development and modulatory role of FGF2 on steroidogenesis and survival of cultured buffalo granulosa cells.

The present study investigated the expression and localization of FGF and its functional receptors in the follicle of buffalo and the treatment of FGF2 on mRNA expression of CYP19A1 (aromatase), PCNA, and BAX (BCL-2 associated X protein) in cultured buffalo granulosa cells (GCs). Follicles were classified into four groups based on size and E2 level in follicular fluid (FF): F1, 4-6mm diameter, E2<0.5ng/ml of FF; F2, 7-9mm, E2=0.5-5ng/ml; F3, 10-13mm, E2=5-40ng/ml; F4, >14mm, E2>180ng/ml. The qPCR studies revealed that the mRNA expression of FGF1, FGF2 and FGF7 were maximum (P<0.05) in theca interna (TI) whereas the transcripts of FGFR1, FGFR2, FGFR2IIIB and FGFR2IIIC were up-regulated (P<0.05) in GCs of F4 follicles. Protein expression of most members were maximum (P<0.05) in F4 follicles except FGFR3 and FGFR4. All members were localized in GC and TI with a stage specific immunoreactivity. Primary culture of GCs with treatment of FGF2 at different dose-time combinations revealed that the mRNA expression and immunoreactivity of CYP19A1 and PCNA were maximum (P<0.05) whereas BAX was minimum (P<0.05) with 200ng/ml at 72h of incubation. The findings indicate that FGF family members are expressed in a regulated manner in buffalo ovarian follicles during different stages of development where FGF2 may promote steroidogenesis and GC survival through autocrine and paracrine manner.

Expression and localization of angiopoietin family in corpus luteum during different stages of oestrous cycle and modulatory role of angiopoietins on steroidogenesis, angiogenesis and survivability of cultured buffalo luteal cells.

The objective of this study was to document the expression and localization of angiopoietin (ANGPT) family members comprising of angiopoietin (ANGPT1 and ANGPT2), and their receptors (Tie1 and Tie2) in buffalo corpus luteum (CL) obtained from different stages of the oestrous cycle, and the modulatory role of ANGPT1 and ANGPT2 alone or in combinations on progesterone (P4 ) secretion and mRNA expression of phosphotidylinositide-3kinase-protein kinase B (PI3K-AKT), phosphoinositide-dependent kinase (PDK), protein kinase B (AKT), Bcl2 associated death promoter (BAD), caspase 3 and von willebrand factor (vWF) in luteal cells obtained from midluteal phase (MLP) of oestrous cycle in buffalo. Real-time RT-PCR (qPCR), Western blot and immunohistochemistry were applied to investigate mRNA expression, protein expression and localization of examined factors whereas, the P4 secretion was assessed by RIA. The mRNA and protein expression of ANGPT1 and Tie2 was maximum (p < .05) in mid luteal phase (MLP) of oestrous cycle. The ANGPT2 mRNA and protein expression was maximum (p < .05) in early luteal phase, decreased in MLP and again increased in late luteal phase of oestrous cycle. ANGPT family members were localized in luteal cells and endothelial cells with a stage specific immunoreactivity. P4 secretion was highest (p < .05) with 100 ng/ml at 72 hr when luteal cells were treated with either protein alone. The mRNA expression of PDK, AKT and vWF was highest (p < .05) and BAD along with caspase 3 were lowest (p < .05) at 100 ng/ml at 72 hr of incubation period, when cultured luteal cells were treated with either protein alone or in combination. To conclude, our study explores the steroidogenic potential of angiopoietins to promote P4 secretion, luteal cell survival and angiogenesis through an autocrine and paracrine actions in buffalo CL.

Expression and localization of angiopoietin family in buffalo ovarian follicles during different stages of development and modulatory role of angiopoietins on steroidogenesis and survival of cultured buffalo granulosa cells.

The present study investigated the expression and localization of angiopoietin (ANPT) family members in buffalo ovarian follicles of different size. It also looked at the role of ANPTs in estradiol secretion and mRNA expression of phosphoinositide-3-kinase-protein kinase B signaling pathway cellular proliferation (phosphoinositide-dependant kinase and protein kinase B [AKT]) and proapoptotic (BAD) factors with caspase 3 in cultured buffalo granulosa cells (GCs). The mRNA and protein expression of ANPT-1 was greatest (P < 0.05), whereas ANPT-2 was reduced (P < 0.05) in preovulatory follicles as compared to F1 follicle. Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 transcripts and protein expression did not change in all follicular groups, whereas tyrosine kinase with immunoglobulin-like and EGF-like domains 2 mRNA was highest (P < 0.05) in theca interna but not GC layer of preovulatory follicle. All members of ANPT family were localized in GC and theca interna showing a stage specific immunoreactivity. Cultured GCs were treated with ANPT-1 and ANPT-2 separately at doses of 1, 10, and 100 ng/mL and in combination at 100 ng/mL for three incubation periods (24, 48, and 72 hours). Estradiol secretion was highest (P < 0.05) at 100 ng/mL at 72 hours of incubation when GCs were treated with either protein alone. The mRNA expression of phosphoinositide-dependant kinase and AKT was highest (P < 0.05), and BAD with caspase 3 was lowest (P < 0.05) at 100 ng/mL at 72 hours of incubation, when cultured GCs were treated separately with each protein or in combination. The immuoreactivity of AKT, pAKT, and pBAD were maximal, whereas BAD was minimal with 100 ng/mL at 72 hours when cultured GCs treated with either protein alone. The findings indicate that ANPTs are expressed in a regulated manner in buffalo ovarian follicle during different stages of development where they may promote steroidogenesis and GC survival through autocrine and paracrine actions.

Modulatory role of leptin on ovarian functions in water buffalo (Bubalus bubalis).

The aim of the present study was to demonstrate the modulatory role of leptin on bubaline granulosa cells (GCs) and luteal cells (LCs) functions using an in vitro cell culture system and to establish a cross talk between leptin and insulin-like growth factor-1 (IGF-1). GCs were collected from group IV follicles (>13 mm size) and LCs from mid-luteal phase corpus luteum and were grown in serum-containing media supplemented with leptin at three different dose rates (0.1, 1, and 10 ng/mL) and time durations (24, 48, and 72 hours). We evaluated the production and secretion of estradiol (E2) and progesterone (P4) using RIA and the mRNA expression of steroidogenic acute regulatory protein (STARD1), cytochrome P450 cholesterol side-chain cleavage (CYP11A1), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 aromatase (CYP19A1), sterol regulatory element-binding protein 1 (SREBP1), steroidogenic factor-1 (SF1), anti-apoptotic gene PCNA, pro-apoptotic gene caspase 3 and endothelial cell marker, Von Willebrand factor (vWF), using quantitative real-time polymerase chain reaction. The results depicted a direct inhibitory action of leptin on GCs steroidogenesis in a time-dependent manner (P < 0.05), whereas in the presence of IGF-1 the inhibitory effect was reverted. Furthermore, leptin augmented both cellular proliferation (PCNA) and apoptosis (caspase 3). On the other hand, in LCs, leptin alone showed an apparent stimulatory effect on steroidogenesis (P < 0.05); however, in the presence of IGF-1, an antagonistic effect was witnessed. Moreover, leptin had an inhibitory effect on apoptosis while promoted cellular proliferation and angiogenesis. These findings were further strengthened by immunocytochemistry. To conclude, these observations for the first time reported that in buffaloes leptin has a direct dose-, time-, and tissue-dependent effect on ovarian steroidogenesis, angiogenesis, and cytoprotection, and furthermore, it can regulate the effect of systemic factors like IGF-1. Hence, this in vitro study provides an insight into the putative roles of leptin alone and its interactions in vivo.

CD10-a new prognostic stromal marker in breast carcinoma, its utility, limitations and role in breast cancer pathogenesis.

BACKGROUND AND AIMS: Breast cancer is one of the leading causes of mortality in Indian women. Although breast cancer is an epithelial malignancy, stroma plays a key role in its development and pathogenesis. Stromal markers are now emerging as novel markers in assessing the prognosis of invasive breast cancer and have not been studied extensively till date. The aim of the present study is to study the stromal expression of CD10 in breast carcinoma, find its relationship with other prognostic markers and study the role stroma plays in breast cancer pathogenesis. MATERIALS AND METHODS: A total of 70 cases of breast cancer were included in the study. Representative sections were taken and hematoxylin and eosin staining was done. Immunohistochemistry was performed with ER, PR, Her2neu and CD10. Stromal expression of CD10 (>10% stromal positivity was considered positive) in invasive breast carcinoma was noted and was statistically analyzed with different known prognostic markers of breast carcinoma. RESULTS: Stromal expression of CD10 was found to be significantly associated with increasing tumor grade (P = 0.04), increasing mitotic rate (P = 0.33), worsening prognosis (P = 0.01), ER negativity (P = 0.0001), Her2neu positivity (P = 0.19) and with molecular subtypes (CD10 positivity with the HER2 type, and CD10 negativity with Luminal type). No correlation was found between CD10 overexpression and PR, age, menopausal status, tumor size, lymph node positivity and tumor stage. CONCLUSIONS: This study gives substantial proof to the various models/research papers explaining the role of stroma/CD10 in breast cancer pathogenesis. Keeping the role stroma plays in predicting prognosis and tumor response, CD10 should be included as a routine pre-chemotherapy marker in breast carcinoma. Further studies should be performed to see the role stroma plays in hormonal expression and the usefulness of CD10 to predict treatment failure in breast carcinomas receiving neoadjuvant therapy.

Awareness and knowledge on eye donation among university students.

Four hundred (400) students studying first year Medicine, Dentistry, Laboratory Technology, Pharmacy, Biomedicine and Bioengineering degree courses in the University of Malaya were assessed on their awareness and knowledge on eye donation using an open ended questionnaire. The majority of the students (344, 86%) in this study were aware about eye donation; the awareness was higher in biomedical (77.1%) and medical students (76.7%) compared to the others (55.9%-70.7%). One hundred and eight students (27%) were willing to donate their eyes. Most of the students (376, 94%) did not know about any eye bank in Malaysia. One hundred and sixty (40%) students were aware that whole eye can be removed from the donor and 101 (25.25%) were aware that the cornea can be removed separately. However, only 121 (30.25%) knew that donated eyes were used for corneal grafting. More than half of the students (231, 57.7%) did not know that the donor eye could be stored before transplantation. The results of this study indicate that there is a need to educate the young adults in our society about corneal transplantation so that they can in turn motivate other members of society and their own family members to become eye donors, thus facilitating the availability of donor corneas for corneal transplantation in Malaysia.

Clastogenicity of carbazole in mouse bone marrow cells in vivo.

Clastogenicity of carbazole was evaluated by employing mouse in vivo chromosomal aberration (CA) test. Carbazole administered intraperitoneally (i.p.) at the rate of 25, 50, 100, 150 and 200mg/kg b.w. to Swiss albino mice in vivo resulted in mitotic depression and induction of chromosomal aberrations. Dose related decrease in mitotic index (MI) and increase in the frequencies of chromosomal aberrations per cell (CAs/cell) and percent abnormal cells were recorded in bone marrow cells. However, statistically significant reduction in MI and increase in CAs/cell and percent abnormal cells were found only for the two higher doses. The results obtained indicate that carbazole or its metabolite, if any, is moderately clastogenic in the bone marrow cells of Swiss albino mice.

Further observations on the nocturnal oviposition behaviour of blow flies (Diptera: Calliphoridae).

Calculating the age of immature stages of blow flies showing the longest period of association with a dead body often gives a fairly accurate estimate of the post-mortem interval (PMI). Determination of the exact time of oviposition by these flies had generally been made in the light of the conventional belief that blow flies are neither active nor do they lay eggs during night. This method of estimating the time of oviposition was modified when Greenberg [J. Med. Entomol. 27 (1990) 807] reported nocturnal oviposition by three calliphorid species that are occasionally used as forensic indicators. However, a technical problem with his experiment, having long term consequences, was placement of the bait on the ground among bushes. This could have made it possible for the flies already resting near the bait to climb over the piece of meat and lay eggs. Though Greenberg's experiment proves beyond any doubt that blow flies do lay eggs at night as well as by day, active attraction of these flies at night towards the oviposition medium had yet to be proved and the present experiment has been designed for this purpose.

Germ cell mutagenicity of phthalic acid in mice.

Mutagenicity of phthalic acid was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice received a single intraperitoneal (i.p.) injection of either 40 mg or 80 mg/kg b.w. of phthalic acid for 5 consecutive days. For the sperm head abnormality assay, the mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w. as a single i.p. injection. Treatment of adult male mice with phthalic acid resulted in induction of dominant lethal mutations and abnormal sperm heads. The results obtained indicate that phthalic acid is a germ cell mutagen.