Immature Research Articles

Abstract A015: Tissue MUC5AC expression predicts recurrence patterns following resection in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) has consistently poor outcomes, even for early-stage tumors that undergo resection (>70%), despite completion of prescribed perioperative systemic therapy. Mucin 5AC (MUC5AC), a heavy glycoprotein, exists in two major glycoforms: heavily glycosylated mature (MM) and less glycosylated immature (IM) forms. MM is located in the apical region and extracellular space, whereas IM is in the perinuclear region. Our previous work demonstrated the association between MM and treatment response and outcomes in resected PDA after neoadjuvant therapy (NAT). In this study, we investigate the impact of MUC5AC tissue expression in resected PDA samples on recurrence site characteristics (local (LR) vs. distant metastasis (mets) vs. both LR and mets). Resected PDA formalin-fixed paraffin-embedded tissue blocks from January 2010 to June 2021 were obtained from the Ohio State University biorepository. Immunohistochemistry was performed to study the expression for MM and IM using monoclonal antibodies 45M1 and CLH2, respectively, and H-scores (0-300) were calculated. Logistic regression was used to study this association of interest. We had 100 R-PDA (43 received NAT, and 57 had upfront surgery (UpS)) available for testing. The median age of diagnosis was 65 years, and 50% were males. Most of the patients in the NAT group received FOLFIRINOX (n=36) and received gemcitabine/nab-paclitaxel (n=5) or FOLFOX (n=2). The median number of doses received is 6 (range 2-9). The results in these groups can be summarized as follows: a) 35/43 had recurrence (LR=7, mets=21, and LR+mets=7), with the liver being the most common site of metastasis (13/28) followed by lung (5/28) and peritoneum (5/28), b) Higher IM expression (mean H-score) was significantly (p=0.0275) higher in LR+mets (H-score 196) than in LR (H-score 69) or mets (H-score 115) groups, c) Higher MM expression (mean H-score) was also significantly different among these groups (LR vs. mets vs. LR+mets = 81 vs. 120 vs. 206, p= 0.0358), e) similar trend of mean H-scores was noted in FOLFIRINOX sub-group (LR vs. mets vs. LR+mets = 205 vs. 109 vs. 60, p=0.0185 for IM and 65 vs. 116 vs. 205, p=0.0329 for MM). No significant association between recurrence patterns and tissue MUC5AC expression in the UpS group. These findings concur with our prior observations, underscoring the clinical significance of MUC5AC in resected PDA post-NAT and suggesting its potential role in facilitating pancreatic tumor cell metastasis. Although larger prospective studies are needed to confirm these results, our study highlights MUC5AC as a potential therapeutic target to prevent metastasis in early-stage PDA, potentially improving overall outcomes. Citation Format: Ashish Manne, Lianbo Yu, Ashwini Esnakula. Tissue MUC5AC expression predicts recurrence patterns following resection in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A015.

Extracellular (EC) Mucin5AC (MUC5AC) detection after neoadjuvant therapy (NAT) and the risk of recurrence in resected pancreatic ductal adenocarcinoma (R-PDA).

4171 Background: The clinical significance of MUC5AC glycoforms is unclear to date. Our earlier study showed the association of lower mature (MM) and immature (IM) MUC5AC in R-PDA with pathological treatment response and EC detection of MM (EC-MM). Here, we have the significance of EC-MM detection in the tissue of R-PDA post-NAT. Methods: Formalin-fixed paraffin-embedded tissue blocks of R-PDA from January 2010 to June 2021 were obtained from the Ohio State University biorepository. Using monoclonal antibodies for 45M1 (MM) and CLH2 (IM), immunohistochemistry was performed to study the cellular expression of MM and IM, and H-scores were calculated. Also, the presence or absence of EC expression of MM is noted. progression-free survival (PFS) and overall survival (OS) were the primary endpoints. Univariate and multivariate Cox regression models were used to model PFS and OS, and clinical and pathological variables were adjusted in the multivariate models. We performed univariate logistic regression analysis to examine the impact of MUC5AC expression on pathological features in resected specimens. Results: Among 100 R-PDA patients, 43 received NAT and 57 had upfront surgery (UpS)). In NAT-group (36-FOLFIRINOX, 2-FOLFOX, and 5-gemcitabine/nab-paclitaxel), EC-MM detection in the resected specimens was associated with worse PFS on multivariate analysis (MVA) (Hazard Ratio (HR) 0.2, 95 % CI: 0.059-0.766, p = 0.01). In the FOLFIRINOX-treated group, the EC-MM detection had a similar impact (HR 0.2, 95 % CI: 0.052-0.847, p = 0.02) on PFS. Cellular MM and IM expression were significant (p < 0.05) on MVA for OS in both groups, but the HRs were not clinically significant (NAT-group, MM - 0.9 & IM - 1.04, FOLFIRNIOX-group, MM - 0.8 & IM -1.02). Expression levels of cellular MM and IM in FOLFIRINOX group and MM alone in NAT-group significantly (p < 0.05) correlated with incomplete resection (R1/R2 vs R0) and perineural invasion. In the UpS group, MUC5AC glycoforms did not significantly correlate with survival or pathological features. Conclusions: Post NAT EC-MM detection and cellular MUC5AC (MM and IM) expression levels are associated with worse PFS and pathological features. Their role in the treatment of naïve patients is still unclear. MUC5AC can be a clinically useful predictive biomarker. Future research should focus examining its importance in advanced tumors and manipulating it for therapeutic purposes.

Differential Expression and Diagnostic Value of MUC5AC Glycoforms in Pancreatic Ductal Adenocarcinoma.

We explored the differential expression and diagnostic value of two significant Mucin 5AC (MUC5AC) glycoforms, less-glycosylated immature (IM) and heavily-glycosylated mature (MM), in neoplastic diseases (NpD), including pancreatic ductal adenocarcinoma (PDA) and neuroendocrine tumors (NET), and non-neoplastic (non-NpD) diseases. Commercially available tissue microarray (TMA) was constructed from 96 patients, including 38 primary PDA (PT), 5 metastatic lesions (ML), 11 NET, and the rest being non-NpD tissues. Immunohistochemistry for MUC5AC was performed using CHL2 and 45M1 clones for IM and MM isoforms, respectively. MUC5AC (both glycoforms) are not detected in non-NpD. In MUC5AC-positive neoplastic tissues, IM was localized to the cytoplasm (Cy) while MM was identified in apical (Ap) and extracellular (Ec) regions too. One ML positive (omentum) in the TMA expressed both. For PDA vs. non-PDA, the sensitivity (SN) was higher with MM ± IM (71%) than MM (47%) or IM (65%)-alone. The specificity (SP) was 100% with MM-alone, which dropped with the addition of IM (96%) or IM-alone (93%). For NpD vs. non-NpD, the SN (MM + IM-59%, IM-55%, MM-37%) was inferior, and SP was 100% for both glycoforms (MM ± IM). The combination of MUC5AC glycoforms has high SP and reasonable SN to diagnose PDA. They have the potential to be a reliable diagnostic marker and should be investigated further in more extensive studies.

Expression profiles of MUC5AC glycoforms in neoplastic and non-neoplastic pancreatic tissue.

e16008 Background: MUC5AC, secretory mucin, exists in two major glycoforms, heavily-glycosylated mature (MM) and less-glycosylated immature (IM) isoforms. Prior IM-based tissue expression studies could not establish the clinical significance of MUC5AC in pancreatic ductal adenocarcinoma (PDA). Despite having strong pre-clinical evidence of MM’s role in local invasion, metastasis, and drug (gemcitabine) resistance, its impact on PDA outcomes is unclear. In this study, for the first time, we examined the differential expression of MM and IM by immunohistochemistry in primary PDA (pPDA), metastatic PDA (mPDA), normal pancreatic tissue adjacent to tumor (NAJ), neuroendocrine tumors (NET), Pancreatitis (P), normal pancreas (N) using tissue microarrays (TMA). Methods: Commercially available TMA (PA2081c, tissuearray.com, Derwood, MD) constructed from 96 patients, including 38 pPDA (14-stage 1, 22-Stage 2, and 2-stage 3), 5 mPDA, 24 NAJ, 11 NET, 8 P and 10 N was used in this study. Two separate 1 mm cores represented each case. Immunohistochemistry for MUC5AC was performed using CHL2 clone (ab77576, abcam, Boston, MA) for the IM isoform and 45M1 clone (ab369, abcam, Boston, MA) for the MM isoform. The immunostains were optimized as per the manufacturer's recommendations using appropriate controls. The sections are evaluated for the intensity of expression (0-3) and the percentage of reactive cells, and an H-score is derived from the product of these measurements. Cases are categorized as having negative/weak (score <10) or positive (score >10) expression. For every positive case, the pattern of expression in terms of Apical (Ap), cytoplasmic (Cy), and extracellular (EC) expression was recorded. Results: MUC5AC (MM and IM) is not expressed in N, NAJ, and P. MM expression is only noted in 44% of PDAs (19/43; 18 pPDA and 1 omental mPDA lesion). Three different expression patterns were noted, including Ap (63%), Cy (84%), and EC (47%). IM expression was detected in 60% of PDAs (26/43, 25 pPDA and omental mPDA lesion) and 18% NET (2/11). IM expression was noted in cases with EC MM expression (9/43, 21%), suggesting that IM was further glycosylated to MM and expressed extracellularly in some patients. MM and MM were co-expressed in 40% of PDA (17/43). Liver and abdomen metastatic lesions in the TMA did not express IM or MM. Pertinent clinical information was not available to study the impact of MM’s expression on PDA outcomes. Conclusions: Our study shows that MUC5AC is primarily expressed in PDAs and can be a useful biomarker. Larger studies are ongoing to correlate tissue expression patterns in PDAs for evidence of any association with early recurrence or treatment failure. Preliminary data suggests that only MM isoform is expressed extracellularly and could be detected in patient serum and serve as a potential non-invasive biomarker for treatment selection (for gemcitabine resistance) and monitor the response in certain cases.

Ultrasound-Assisted Extraction of Phenolic Compounds from Different Maturity Stages and Fruit Parts of Cordia dodecandra A. DC.: Quantification and Identification by UPLC-DAD-ESI-MS/MS

In the present work, the total phenolic content (TPC), total flavonoids content (TFC), antioxidant activity, and phenolic profile from pulp (PU) and peel (PE) extracts obtained from the ciricote (Cordia dodecandra A. DC.) fruit by ultrasound-assisted extraction (UAE) in immature (IM), semimature (SM), and mature (MM) stages were investigated. The effect of the diameter of the ultrasonic probe in the IM stage was also evaluated. The TPC and antioxidant activity in IM fruit extracts by UAE increased up to 11.01 and 23.82 times, respectively, compared to the maceration method. The main phenolic compounds in the PE of IM fruit identified by UPLC-DAD-ESI-MS/MS were quantified as caffeic acid, rutin, and rosmarinic acid, distributed as 45.82, 41.45, and 12.72%, respectively. The PE extracts of IM fruit obtained with the 3 mm diameter probe had 1.27, 2.44, and 1.37 times the TPC (19.93 ± 0.28 mg GAE (Gallic equivalents) g−1 dw), TFC (34.85 ± 4.99 mg RE (Rutin equivalents) g−1 dw), and antioxidant activity (122.09 ± 17.09 µTE (Trolox equivalents) g−1 (DPPH)), respectively, compared to those obtained with a 13 mm diameter probe. The results obtained suggest the use of the ciricote native fruit as a source of bioactive compounds, directly as fresh fruit or processed, thus helping to increase its production and consumption.

Anterior Vertebral Body Tethering for Scoliosis Patients With and Without Skeletal Growth Remaining: A Retrospective Review With Minimum 2-Year Follow-Up.

Anterior vertebral body tethering (AVBT) has been approved for skeletally immature (IM) adolescent idiopathic scoliosis patients, but the role of AVBT in patients with minimal remaining skeletal growth is controversial. The purpose of this study was to compare minimum 2-year (YR2) outcomes in skeletally IM patients vs those with minimal remaining skeletal growth. Patients with single thoracic AVBT were grouped by their preoperative (PR) skeletal maturity: IM (n = 16, Risser 0-2) vs mature (M, n = 19, Risser 3-5). Outcomes were assessed at PR, first erect (FE), and YR2. Median (range) was compared with nonparametric tests (P < 0.05). The PR age was 12.5 (9-16) vs 15 (12-18) years with major Cobb 51° (36°-69°) and 49° (40°-69°) for IM and M, respectively. At FE, there was no difference in correction; however, at YR2, the IM group yielded a lower residual curve (15° [-16° to 38°] vs 29° [12°-42°], P = 0.008). Thoracolumbar/lumbar curves were corrected without group differences. Clinically successful correction (<35°) (15 [94%] vs 15 [79%]) and suspected cord breakages (2 [13%] vs 2 [12%]) were similar at YR2. Two overcorrections occurred, both in IM patients. Scoliosis Research Society-22 outcomes at final follow-up were similar between groups. No revision reoperations or conversions to spinal fusion were needed. Skeletally IM patients benefit from greater growth-modulated curve correction than M patients, however, at the increased risk of overcorrection. M patients maintained clinically significant correction at latest follow-up. Longer-term follow-up is required to determine durability of outcomes for patients undergoing AVBT who have minimal remaining growth at the time of index surgery. This study is relevant to spine surgeons, spine physiotherapists, and patients with idiopathic scoliosis. It offers evidence of clinical correction of scoliosis in mature patients.

Comparative liver transcriptome analysis of duck reveals potential genes associated with egg production.

Molecular studies on egg production in ducks were mostly focused on brain and ovaries as they are directly involved in egg production. Liver plays a vital role in cellular lipid metabolism. It also plays a decisive role in reproductive organ development, including yolk generation in laying ducks at sexual maturity. However, the precise molecular mechanism involved in the liver-blood-ovary axis in ducks remains elusive. In this study, we analysed the liver transcriptome of laying (LA), immature (IM) and broody (BR) ducks using RNA sequencing to understand the role of genes expressed in the liver. The comparative transcriptome analysis revealed 82 DEGs between LA and IM ducks, 47 DEGs between LA and BR ducks and 51 DEGs between IM and BR ducks. GO analysis of DEGs, showed that DEGs were mainly involved in cellular anatomical entity, intracellular, metabolic process, and binding. Furthermore, pathway analysis indicated the important role of Wnt signaling pathway in egg formation and embryo development. Our study showed several candidate genes including vitellogenin-1, vitellogenin-2, riboflavin binding protein, G protein subunit gamma 4, and fatty acid binding protein 3 that are potentially related to egg production in ducks. The study provides valuable information on the genes responsible for egg production and thus, pave the way for further investigation on the molecular mechanisms of egg production in duck.

Identification of regulatory factors promoting embryogenic callus formation in barley through transcriptome analysis

BackgroundBarley is known to be recalcitrant to tissue culture, which hinders genetic transformation and its biotechnological application. To date, the ideal explant for transformation remains limited to immature embryos; the mechanism underlying embryonic callus formation is elusive.ResultsThis study aimed to uncover the different transcription regulation pathways between calli formed from immature (IME) and mature (ME) embryos through transcriptome sequencing. We showed that incubation of embryos in an auxin-rich medium caused dramatic changes in gene expression profiles within 48 h. Overall, 9330 and 11,318 differentially expressed genes (DEGs) were found in the IME and ME systems, respectively. 3880 DEGs were found to be specific to IME_0h/IME_48h, and protein phosphorylation, regulation of transcription, and oxidative-reduction processes were the most common gene ontology categories of this group. Twenty-three IAA, fourteen ARF, eight SAUR, three YUC, and four PIN genes were found to be differentially expressed during callus formation. The effect of callus-inducing medium (CIM) on IAA genes was broader in the IME system than in the ME system, indicating that auxin response participates in regulating cell reprogramming during callus formation. BBM, LEC1, and PLT2 exhibited a significant increase in expression levels in the IME system but were not activated in the ME system. WUS showed a more substantial growth trend in the IME system than in the ME system, suggesting that these embryonic, shoot, and root meristem genes play crucial roles in determining the acquisition of competency. Moreover, epigenetic regulators, including SUVH3A, SUVH2A, and HDA19B/703, exhibited differential expression patterns between the two induction systems, indicating that epigenetic reprogramming might contribute to gene expression activation/suppression in this process. Furthermore, we examined the effect of ectopic expression of HvBBM and HvWUS on Agrobacterium-mediated barley transformation. The transformation efficiency in the group expressing the PLTPpro:HvBBM + Axig1pro:HvWUS construct was increased by three times that in the control (empty vector) because of enhanced plant regeneration capacity.ConclusionsWe identified some regulatory factors that might contribute to the differential responses of the two explants to callus induction and provide a promising strategy to improve transformation efficiency in barley.

Assessment of compounds and cytotoxicity of Citrus deliciosa Tenore essential oils: From an underexploited by-product to a rich source of high-value bioactive compounds

Essential oil (EO) from the peels of Citrus deliciosa fruits are underexploited among Citrus EO by the food and cosmetic industries. The aims of this study were to evaluate the biological effects of EO from C. deliciosa fruit peels at different stages of maturation in an adenocarcinoma cell line and propose the use of this by-product as a new source for the extraction of bioactive compounds. The fruits were collected at the immature (IM), intermediate (INT), and mature (MAT) stages. A total of 15, 17, and 14 components were measured using GC-MS in the EO at the IM, INT, and MAT stages, respectively. The main components were limonene and γ-terpinene at all the stages. The in vitro antioxidant activity was evaluated using ABTS+• assay, and EO INT showed the greatest activity with an IC50 value of 0.264 ± 0.004 mg/mL. The cytotoxic effect was evaluated against HT-29 cells, in which EO MAT showed significant cytotoxic activity with an IC50 of 110 ± 10 μg/mL. It was found that the presence of citronellol and β-linalool favors the antioxidant activity; however, their presence suggested a decrease in the cytotoxic activity, when associating the phytochemical profile with the evaluated activities. The results showed that secondary compounds at trace concentrations were associated with differences in biological activities. C. deliciosa fruit peel, a by-product of food juice industry, might be used as a source for the extraction of bioactive compounds with promising biological activities.

P14.50 Clinicopathological spectrum of intracranial germ cell tumors: an Indian tertiary cancer center experience

Abstract BACKGROUND Incidence of intracranial germ cell tumors (ICGCT) in Western literature is low (0.3–0.6 %) as compared to East Asia (3–4 %), &amp; their clinicopathological features are well documented. However, there are scant studies on ICGCT from India. MATERIAL AND METHODS Retrospective observational study of all ICGCT histologically diagnosed in our hospital from 2007–2018. Metastasis were excluded. Clinicopathological features were retrieved from hospital’s electronic medical records. RESULTS We diagnosed 82 primary ICGCT, forming approx. 0.54 % of all primary brain tumors, &amp; 3.5% of pediatric brain tumors. Age range: 2 months-32 yrs (Median age 14 yrs). M:F ratio: 1.82:1 (53M,29F). Nearly 80% patients were pediatric (&lt;18 yrs), &amp; 8 very young (&lt;3 yrs, 7M1F). Majority were suprasellar &amp; pineal (31/82, 37% each), with one bifocal presentation. Other rarer sites: posterior fossa (4), midbrain (1), corpus callosum (1) &amp; 11 non midline (4 thalamic, 4 frontal, 2 cerebellar, 1 CP angle). Predominant histology was germinoma (G) (51/82, 62%), while non germinomatous (NGGCT) were 31/82 (38%), of which 9 were mixed. Pure teratoma were 11 (9 immature (IT), 2 mature), &amp; 5 pure yolk sac tumor (YST). Interestingly, all very young age group patients (&lt;3 yrs), showed only NGGCT histology (5/8 IT, 3/8 pure YST). In contrast, G histology formed nearly 70% of all patients &gt;3 yrs. Females were associated mainly with G (21/29, 72%). NGGCT were predominantly seen in males (M:F=2.9:1). Also, pure IT (9) were seen only in males. Posterior fossa tumors were all IT (4/4). Spinal tumors were NGGCT (1 mature teratoma, 1 YST). Majority of suprasellar tumors (25/31, 80.6%), other midline locations like corpus callosal, midbrain, &amp; all thalamic tumors were G. However, pineal tumors showed equal distribution of G (15/31) &amp; NGGCT (16/31). Spine screening was positive in 8 patients (6 G, 2 IT)- 7 on MRI and 1 only on CSF cytology. Serum tumor markers were raised in 13/54 cases- 6/34G(17.6%), vs 7/20 NGGCT (35%). CSF tumor markers were raised in 14/34-10/23 G (43.4%), vs 4/11 NGGCT (36.3%). Follow up was available for 37 patients (Duration 3 months-10 yrs, median 2 yrs). On f/u 6/15 (40%) NGGCT showed progression/death, while only 2/32 G relapsed (6.5%). Four deaths in G group were not directly attributable to the tumor. CONCLUSION Frequency of ICGCT in our hospital similar to western data rather than Asian, albeit with less striking male preponderance. ICGCT were tumors of 2nd decade &amp; majority occurred in pineal/suprasellar areas. About 2/3rd were pure G on histology, and showed good prognosis. NGGCT were common in infants, males and in posterior fossa. IT were seen exclusively in males and pure YST mainly in males. Although majority of ICGCT are in midline, rarely non midline involvement also occurs, and it’s essential to exclude metastasis before considering primary ICGCT. Expectedly, NGGCT showed poorer prognosis, compared to pure germinomas.

107 Insights from roe deer oocyte transcriptome across embryonic diapause

Embryonic developmental arrest, known as diapause, has been reported in more than 130 species. However, its mechanisms are still not completely understood. In the roe deer, the only known ungulate that exhibits this phenomenon, diapause lasts for approximately 5 months, starting after the rut period in mid-July to early August and ending with embryo elongation and implantation in December/January. Little is known regarding oocyte characteristics during this period. Here, we analysed the roe deer oocyte transcriptome as a model to understand diapause effects on oocyte features during embryonic developmental arrest and reactivation. During regular hunting, immature oocytes were obtained by ovary slicing from diapause and nondiapause stages, and classified according to morphological characteristics. Only oocytes with &amp;gt;2 layers of compact cumulus cells and cytoplasm from 30 hunted females were used for analyses. Immature oocytes were denuded and snap frozen. Additional oocytes were cultured in maturation medium for 20-24h. Matured oocytes with a present polar body were snap frozen. Two pools of 10 immature and mature oocytes for both diapause and nondiapause stages were included (at least 4 donors/pool). Oocyte pools were processed using the Smart-seq 2 single-cell protocol (Illumina Inc., San Diego, CA, USA) for full-length cDNA and library preparation. We performed RNA-seq on an Illumina sequencer. The obtained Fastq files were clipped and analysed with a locally installed version of the Galaxy platform. Sequences were mapped against the roe deer transcriptome (unpublished data) and annotated against human and bovine transcripts. Differentially expressed genes (DEG; false discovery rate &amp;lt;1%) were identified using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) in immature (IMM) and mature (M) oocytes from diapause (D) and nondiapause (ND) stages. Additionally, to evaluate the effects of maturation on oocyte transcript abundancy, DEG between IMM and M oocytes for D and ND were identified. Multidimensional scaling resulted in clustering according to oocyte types. Gene ontology terms for biological processes were assigned using ToppCluster tools. A total of 23066, 23022, 22438, and 22532 transcripts were detected for IMM D, M D, IMM ND and M ND oocytes, respectively. Using false discovery rate filtering, we found 333 and 288 DEG in immature and mature oocytes, respectively. Furthermore, maturation changed the oocyte expression profiles during D (2233 DEG) and ND periods (2589 DEG). Gene ontology classification revealed that most of the DEG in immature oocytes were involved in macromolecule catabolic process and oxidation-reduction process. For mature oocytes, DEG were mostly related to regulation of chromosome condensation and ER and Golgi vesicle-mediated transport. Additionally, the top 20 DEG from IMM D v. M D oocytes were related to mRNA splicing, mRNA metabolic process, and mRNA processing. These processes were not identified in the top 20 DEG from IMM ND v. M ND oocytes. These preliminary results suggest that oocyte transcriptome analysis could disclose new pathways implicated in oocyte competence and embryonic developmental arrest and activation stages.

Distinct histomorphology for growth arrest and digitate outgrowth in cultivated Haliclona sp. (Porifera: Demospongiae).

The use of sponges in biotechnological processes is limited by the supply problem, and sponge biomass production is becoming a current topic of research. The distinction between characteristics for growth and growth arrest is also important for environmental monitoring. In this study, we analyze the morphology of the digitate outgrowths from the sponge Haliclona sp. The sponge Haliclona sp. was successfully cultivated for 14 months in a closed system. The morphological characterization of growth arrest was performed after submitting explants to starvation-stress for approximately 2 weeks, to correlate morphology with growth and growth arrest. The digitate outgrowth showed three distinct regions: mature (MR), transition (TR) and immature (IR). Our data suggest a growth developmental program, with collagen fascicles guiding axial growth in IR, followed by progressive development of choanocyte chambers and large aquiferous systems at the more mature proximal region (choanosome). The intercalation of choanocyte chambers and small aquiferous systems inside collagen fascicles previously originated at the IR region can be responsible for thickening expansion and conversion of the collagen fascicles into columnar choanosome in MR. The growth arrest after starvation-stress assay showed morphological changes in the IR corroborating collagen in the extreme tip of the digitate outgrowth as an important role in guiding of axial growth of Haliclona sp. The identification of distinct morphologies for growth and growth arrest suggest a growth developmental program, and these data could be useful for further investigations addressing sponge biomass gain and environmental monitoring.

Functional morphology of the male reproductive system in Callichirus major (Crustacea: Decapoda: Axiidea): Evidence of oocytes in the gonad

AbstractCallichirus major inhabits the intertidal region of marine ecosystems and it is frequently used as live bait for fishing. This study aimed to describe the functional anatomy of the male reproductive system by microscopic techniques. The animals were collected along the Corujão beach, Piuma—ES, Brazil, and, in laboratory, the males were classified into two phases: immature (IM) and developed (DE) based on the macroscopic characteristics of the gonads. The gonad and vas deferens were dissected for histological routine and histochemical tests. Histologically, it was noted that in both phases, the more distal region of gonads has ovarian characteristics, showing developing oocytes. Also, different male germ cells were identified: spermatogonium (SPG), spermatocytes I and II (SPTCI, SPTCII), initial and final spermatid (IS, FS) and sperm (SPZ). Accessory cells with spherical or pyramidal nuclei were also present inside the testicular lobules. According to the vas deferens structure, three regions can be characterized: proximal (PVD), middle (MVD) and distal (DVD). In the lumen of the vas deferens, a spermatophoric matrix highly reactive for histochemical tests was observed. The presence of female germ cells in males suggests the occurrence of intersexuality or hermaphroditism in this species.

Identification, characterization and target gene analysis of testicular microRNAs in the oriental fruit fly Bactrocera dorsalis

MicroRNAs (miRNAs) are small noncoding RNAs that regulate various diverse biological processes including insect spermatogenesis. The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive horticultural pests in East Asia and the Pacific region. Although developmental miRNA profiles of B. dorsalis have enriched our knowledge, specific testicular miRNAs in this dipteran species are unexplored. In this study, we identified miRNAs from B. dorsalis testes by deep sequencing, which provided an overview of miRNA expression during spermatogenesis. Small RNA libraries were constructed from the testes of fully mature (FM), immature (IM) and middle-aged (MA) adult flies of B. dorsalis. Small RNA sequencing and data analysis revealed 172 known and 78 novel miRNAs amongst these libraries. Pairwise comparisons of libraries led to the identification of 24, 15 and 14 differentially expressed miRNAs in FM vs. IM, FM vs. MA and IM vs. MA insects, respectively. Using a bioinformatic approach, we predicted 124 target genes against the 13 most differentially expressed miRNAs. Furthermore, the expression patterns of six randomly selected miRNAs (from the 13 most differentially expressed miRNAs) and their putative target genes (from the 124 predicted target genes) were analysed in the testis of B. dorsalis by quantitative real-time PCR, which showed that out of six, four tested miRNAs-mRNAs had an inverse expression pattern and are probably co-regulated. This study is the first comparative profile of the miRNA transcriptome in three developmental stages of the testis, and provides a useful resource for further studies on the role of miRNAs in spermatogenesis in B. dorsalis.

Expression of mRNA Encoding IGF-I, IGF-II, Type-I, and II IGF-Receptors and IGF-Binding Proteins-1-4 during Ovarian Follicular Development in Buffalo (Bubalus bubalis)

The present study was designed to investigate the expression pattern of IGF-I, IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in different stages of buffalo ovarian preantral follicles (PFs), antral follicles (AFs), ovulatory follicles (OFs), and immature (IM) and in vitro matured (MO) oocytes. Buffalo ovaries were collected from local abattoir, PFs (200-250 µm), AFs (1-3 mm), and OFs (5-8 mm) were isolated by mechanical method. PFs, AFs, OFs, and oocytes were lysed to release mRNA, reverse transcribed, and then subjected to RT-PCR, whereas protein were localized through immunohistochemistry. Relative expression of mRNA transcripts was clearly seen for IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in all the stages of developing follicles and oocytes. We were unable to detect mRNA and protein expression of IGF-1 in any of the oocytes or follicles at any stage of the development. IGF-II and both IGF receptors mRNA expression were found higher (P < 0.05) in PFs compared to AFs and OFs. Expression of IGFBP-1 and 2 in PFs, as well as IGFBP-3 and 4 in AFs, was found with higher (P < 0.05) levels. The expression results were further confirmed by localization of IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 proteins. In conclusion, IGF-II appears to be the only ligand that is endogenously expressed by all the follicular stages and oocytes, which may act in an autocrine manner through the Type-1 IGF receptor. Expression of IGFBP-1-4 and IGF-II suggests the possible role of these genes in recruitment, growth, proliferation, and steroidogenic responses during developmental phases of buffalo ovarian follicles.

Characterization of gonadotropin-releasing hormone and gonadotropin in jack mackerel (Trachurus japonicus): Comparative gene expression analysis with respect to reproductive dysfunction in captive and wild fish

To better understand the physiological mechanisms underlying reproductive dysfunction, such as impaired vitellogenesis and final oocyte maturation, we assessed endocrinological differences between captive and wild female jack mackerel (Trachurus japonicus). The presence of three gonadotropin-releasing hormone (GnRH) peptides was immunologically evaluated in jack mackerel brain tissues. Full-length cDNAs encoding GnRHs (gnrh1, gnrh2, and gnrh3) and gonadotropin subunits (fshb, lhb, and gpa) were cloned, sequenced, and quantitatively assayed. Of the captive females, 60% failed to undergo vitellogenesis, displaying immature (IM) or atretic oocytes, whereas 80% of wild females were captured during late vitellogenesis (LV) or ovulation (OV). The gnrh1 expression was significantly lower in captive fish than in wild LV and OV fish, while there were no significant differences in the expression of gnrh2 or gnrh3. The expression of fshb was lower in captive IM fish than in wild LV fish, but no significant differences were observed between the captive IM and LV individuals. The lhb expression was elevated in the wild LV and OV fish, and gpa expression was greatest in the wild OV fish. Serum estradiol-17β levels were significantly lower in captive IM fish than in captive LV fish. The results indicate that captive-rearing stress may impair vitellogenesis and negatively influence the transcription of gnrh1 in the brain and GtH synthesis in the pituitary.

Pseudo-Norwid, czyli co się komu podoba

The present text critically discusses Wiesław Rzońca’s book Premodernizm Norwida – na tle symbolizmu literackiego drugiej połowy XIX wieku (Norwid’s Pre-modernism – Against the Background of the Literary Symbolism of the Second Half of the 19th Century) (Warszawa 2013). In the subsequent chapters the present polemics formulates the following charges against the book: 1). The division of Norwid’s work into “the mature period” and “the imma ture one” is unjustified and devoid of factual foundations; especially when in the allegedly “immature” period (until 1857) a lot of the poet’s undoubted masterpieces were written, also, the work after 1857 continued the earlier forms, ideas and motifs, and in this sense it was characterized by a relative – dynamic, flexible and open to changes – continuity. 2). One of Rzońca’s theses – an exceptionally doubtful one – puts Norwid’s allegedly “mature” works after 1857 outside Romanticism, which differs from the truth, both textual and historicalliterary, because Norwid till the end of his life drew on the Romantic tradition and traces of this practice can be discovered in most of his writings and poems, starting from his lectures On Juliusz Słowacki putting Byron (and not Baudelaire!) in the position of “poets’ Socrates”, and ending with Rzecz o wolności słowa (On the Freedom of Speech), perfectly well analyzed in the past by the late Zofia Stefanowska with respect to the Romantic influences retained in the poem, and with Milczenie (Silence), Ad leones!, Stygmat (Stigma) and Tajemnica lorda Singleworth (Lord Singleworth’s Secret). 3). Rzońca’s false diagnoses concerning Norwid’s relation towards Romantics and Romanticism result from a) accepting an extraordinarily narrow, static and basically false conception of Romanticism, reducing it personally to Mickiewicz and Byron, and ignoring the German, English, French, American, or even Polish Romanticism that was intellectually rich and creative (“not-well-enough-read” Krasiński or Słowacki plus the interesting Polish nationalist philosophy), and on which the whole 19th century, not only Norwid drew; b) the false (and grotesque) assumption that since Norwid drew on the European Romantics ample heritage, he necessarily deserves the strict etiquette of a “Romantic”, and the period of 1848-1857 should be absolutely associated with his “immaturity”. 4). Rzońca’s flagship theses about Norwid’s “pre-modernism” does not meet the conditions of sense, for anything you like may be associated with the concepts of “pre-modernism” or “modernism” that are used in the book. 5). The above charge also concerns the thesis about Norwid’s alleged “modernist symbolism”, as this kind of symbolism appeared only after the death of the poet. 6). Hence if it is possible to ascribe some significant historical-literary discovery to Rzońca, it is probably only the one creating Norwid as a posthumous symbolist. 7). Rzońca’s self-appraisal saying that his book presents “an attempt at a synthesis of Norwid’s mature work” (p. 9) should be assessed not only as an attempt – in an arrogant language, which Rzońca does not hesitate to use towards Norwid – decidedly “immature”, but as an unintentional parody of a “synthesis”. This is because Premodernism is a collection of subjective, not thought through impressions that say a lot about their author and his ambitions, but not much about the real, historical Norwid. Hence the title of the review: Pseudo-Norwid.

Mud Crab (Scylla spp.) Aquaculture in the South-West Sundarbans Region of Bangladesh

Mud crab ( Scylla spp.) aquaculture has become an important livelihood option for the coastal fisher-folks in B angladesh. Mud crabs harvested from wild sources are traditionally fatte ned in pens installed in tidal rivers, ghers , and earthen ponds in Bangladesh. The study was ai med at understanding the practice and production economics of mud crab farming in the South-West Sundarbans region of Bangladesh, and was conducted in the areas namely Bagerhat, Satkhira an d Khulna districts where mud crab fattening are predominately ventured. Most of the farmers fatten both male (lean) and female (gonadally immat ure) crabs of 100 - 180 g. The crabs are stocked at varying rate (12 - 641 g/m 2 ), and fed at the rate of 8-10% of body weight with trash live fishes, dri ed fishes, snails, mollusks, and cattle viscera. The fattening for a mean cycle of 22 ± 14.34 days has the survival rate of 55 - 90%, and has the production of 0.065 ± 0.26, 0.084 ± 1.33, and 0.17 ± 3.76 kg/m 2 in Bagerhat, Satkhira, and Khulna, respectively. After harvesting (mostly part ial), crabs are sold at average price rate of 4.35 ± 0.45 US$/kg. The lowes t production cost (3.14 US$/kg) and the better economic return (US$ 147.77) per cycle was recorded in the district of Satkhira. The mud crab fattening has hitherto

미성숙 목화다래 메탄올 추출물의 항산화 효능 평가

Abstract - The results of the content of total polyphenol and flavonoid, DPPH (1-1-diphenyl-2-picryl-hydrazyl) radical scavenging activity, ABTS [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] radical scavenging activity, nitrite scavenging activity and SOD-like activity of methanol extracts from immature cotton boll are follows. The contents of total polyphenol and flavonoid compound were higher in small size cotton boll, and DPPH and ABTS radical scavenging activity also showed a relatively high activity in the small size. These results indicate that there is a correlation between phenol content and DPPH radical scavenging, ABTS radical scavenging. The test concentrations of imma ture cotton boll extract for measuring DPPH and ABTS radical scavenging activities were se t as 1.25, 2.5, 5, 10 and 20 mg/ml. Immature cotton boll has high radical scavenging activity at the concentration of 1.25~20 mg/ml and the result showed a tendency to increase in a concentration-dependent. The nitrite scavenging activity showed high activity in the pH 1.2, and the result in the pH 4.2 showed progressively less active, and in the pH 6.0 near neutra l was confirmed that does not affect the nitrite scavenging. In addition, SOD-like activity showed somewhat lower activity compared with ascorbic acid, but tended to be higher when compared with the results of the other natural substances. Thro ugh this experiment, we confirmed that immature cotton boll was excellent antioxidant activity. Therefore, it is demonstrated that the cotton suggest the possibility of development of new material for cosmetic product or functional food in the future, and is expected to make a greater usability.

Field Release in Florida ofDiaphorencyrtus aligarhensis(Hymenoptera: Encyrtidae), an Endoparasitoid ofDiaphorina citri(Homoptera: Psyllidae), from Mainland China

The Asian citrus psyllid, Diaphorina citri Kuy awama (Hemiptera: Psyllidae), is a serious pest of citrus that vectors the phloem-limited non culturable bacterium Candidatus Liberibacter asiaticus Jagoueix, Bove and Gamier (Jagoueix et al. 1994). The resulting disease is commonly referred to as citrus greening or huanglongbing (HLB) (Bove 2006). The psyllid was first reported from Florida in 1998 and citrus greening in 2005 (Halbert 1998, 2005). Two species specific parasitic wasps, Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Shafee, Alam and Argarwal) (Hymenoptera: Encyrtidae), were imported into Florida from Taiwan and Vietnam and released beginning in 1999 as biological con trol agents for D. citri (Hoy & Nguyen 1998). Tam arixia radiata has established in Florida (Qureshi et al. 2009), while D. aligarhensis has not. An additional population of D. aligarhensis was collected in Guangdong province, China and sent to Gainesville, Florida in 2006. A colony was initiated in the Maximum Security Room of the Florida Biological Control Laboratory, Florida De partment of Agriculture and Consumer Services, Division of Plant Industry. Parasitoids were held in quarantine through 20 generations before re lease permitting was complete and mass rearing began. Sixty-seven releases were made at 10 sites in 6 Florida counties (Alachua, Collier, DeSoto, Hendry, Orange and Polk) between 8/2007 and 10/2009, deploying a total of approximately 11,100 wasps (Table 1). Sites included commer cial and private citrus groves as well as Murraya paniculata hedges. Wasps were either tapped out of holding tubes directly onto emerging plant shoots infested with immature psyllids or were released into cages consisting of fine mesh bags tied around new shoots infested with nymphs. Shoots were carefully inspected and predators removed prior to tying cages closed. Mesh size al lowed parasitoid wasps to move freely in or out of the cage while preventing most predaceous in sects from entering. Released wasps successfully parasitized nymphs in the field and second generation wasps emerged. Evidence of this includes laboratory emergence of adult wasps from field clippings, D. aligarhensis parasitized mummies found on shoots covered with mesh cages, and several adult wasps sighted foraging on D. citri infested M. paniculata plants (Table 1). All citrus groves used for wasp release, including certified organic groves, used pesticide sprays dur ing the release period. The use of insecticides target ing D. citri likely reduced both psyllid and natural enemy populations (Qureshi & Stansly 2007). Mur raya paniculata release sites were not subjected to any form of insect control, although occasional plant trimming by the property owners removed much of the new soft growth that I). citri uses for feeding and oviposition, consequently leaving few imma ture hosts for D. aligarhensis reproduction. Adult psyllids were present at all locations throughout the release period although their incidence appeared to decline over the >2 year release period, likely from increased insecticide use to reduce the spread of citrus greening dis ease. During many trips to various locations for parasitoid release there was little to no host plant flush or immature psyllids present, particularly during late summer and fall. Tamarixia radiata wasps were present at all release sites utilized in this study. Through the combined effect of host feeding and parasitism, a single female T. radiata can kill nearly twice as many psyllids in her lifetime as can a female D. aligarhensis (500 vs. 280, respectively) (Chien 1995). Additionally, T. radiata has a shorter gen eration time and a higher reproductive rate (Skel ley & Hoy 2004). Furthermore, any hosts parasit ized by T. radiata before or within 5 d following parasitism by D. aligarhensis will result in T. radiata offspring (E. R. unpublished data). Thus, competition with T. radiata could present another impediment to establishment of D. aligarhensis. Coccinellid predators were seen at all release sites. Generalist predators can induce high lev els of psyllid mortality (Qureshi & Stansly 2009). Curinus coeruleus Mulsant, Cycloneda sanguinea (L.) and Harmonia axyridis Pallas readily con sumed D. citri nymphs parasitized by D. aligar hensis (E. R. unpublished data) causing wasp mortality.