To establish an untransfected human corneal endothelial (HCE) cell line and characterize its biocompatibility to denuded amniotic membrane (dAM).Primary culture was initiated with a pure population of HCE cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various supplements. The established cell line was characterized by growth property, chromosome analysis, morphology recovery, tumorigenicity assay, and expression of marker proteins, cell-junction proteins, and membrane transport proteins. The biocompatibility of HCE cells to dAM was evaluated by light microscopy, alizarin red staining, immunofluorescence assay, and electron microscopy.2.HCE cells proliferated to confluence 6 weeks later in primary culture and have been subcultured to passage 224 so far. A continuous untransfected HCE cell line with a population doubling time of 26.20 h at passage 101 has been established. Results of chromosome analysis, morphology, combined with the results of expression of marker protein, cell-junction protein and membrane transport protein, suggested that the cells retained HCE cell properties and potencies to form cell junctions and perform membrane transport. Furthermore, HCE cells, without any tumorigenicity, could form confluent cell sheets on dAMs. The single layer sheets that attached tightly to dAMs had similar morphology and structure to those of HCE in situ and had an average cell density of 3,413±111 cells/mmAn untransfected and non-tumorigenic HCE cell line has been established, and the cells maintained positive expression of marker proteins, cell-junction proteins and membrane transport proteins. The cell line, with excellent biocompatibility to dAM, might be used for in vitro reconstruction of HCE and provides a promising method for the treatment of diseases caused by corneal endothelial disorders. The dencapacity .HCE cell lines could provide efficient models for studies of cellular specification, cellular signaling, cell replacement, in vitro reconstruction of tissue-engineered HCE (TE-HCE), immunology of HCE graft rejection, and molecular pathways regulating normal HCE cell homeostasis ,8. The m+/K+-ATPase, voltage-dependent anion channels (VDACs), chloride channel proteins (ClCs), sodium bicarbonate cotransporter 4 (NBC1), and cystic fibrosis transmembrane conductance regulator (CFTR) [Since no definitive markers for HCE cells have been identified, HCE cells can only be characterized with various marker proteins such as neuron specific enolase (NSE), type IV collagen, and vascular endothelial growth factor receptor II (FLK-1), various cell-junction proteins such as zonula occludens protein 1 (ZO-1), N-cadherin, connexin 43 and integrin αv/β5, and various membrane transport proteins such as aquaporin 1 (AQP1), Nar (CFTR) ,12-18. TCorneas from a woman (26 years old) who died due to cerebral hemorrhage were obtained from The Affiliated Hospital of Medical College, Qingdao University , with permission from her next of kin. The usage of the corneas as the source of HCE cells for in vitro culture was approved by the Medical Ethics Committee of the hospital and the privacy of the patient was protected in compliance with the Declaration of Helsinki. SPF BalB/c nude mice , weighing 18 to 22 g, were purchased from Slaccas Experimental Animal Co., Ltd. . All animals were treated in accordance with the ARVO Statement for usage in tumorigenesis assay, and were approved by the Clinical Research Ethics Committee of New Drug Evaluation Center, Shandong University .2. During logarithmic phase, the culture supernatant of HCS cells was collected and centrifuged at 2,000× g for 30 min.Fresh amniotic membranes (AMs), stored in −20 °C glycerol, were obtained from Shandong Eye Institute of Shandong Medical Academy. The AMs were washed twice with 0.9% physiologic saline and disinfected with 1,000 IU/ml tobramycin for 20 min. After a further wash with physiologic saline, the AMs, with the epithelial surface down, were spread uniformly without folds on filter papers fully immersed in 0.02% EDTA-0.25% trypsin  solution. After digestion at 37 °C for 30 min, AMs were washed and their epithelial surfaces were scraped gently with a sterile cell-scraper to obtain denuded AMs (dAMs). Human corneal stromal (HCS) cells, from an HCS cell line established in our laboratory, were cultured in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F12  medium  containing 20% bovine calf serum  at 37 °C with 5% CO2. The corneal pieces were detached and re-attached sequentially into new gelatin-coated wells for a time interval of 36 h, 24 h, 12 h and 6 h, respectively. The wells containing only a pure population of HCE cells were selected, and the cells in these wells were collected. The pure HCE cells were resuspended in 1 ml of 20% FBS-containing DMEM/F12 medium with antibiotics, and split into two new gelatin-coated wells and cultured at 37 °C with 5% CO2. Twenty-four h later, the culture medium was further supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 10 ng/ml epidermal growth factor (EGF), 50 μg/ml N-acetylglucosamine hydrochloride, 50 μg/ml glucosamine hydrochloride, 0.8 mg/ml chondroitin sulfate, 50 μg/ml oxidation-degradation products of chondroitin sulfate , 50 μg/ml carboxymethyl-chitosan , 5 ng/ml bovine ocular extracts , and 10% (v/v) culture supernatant of HCS cells at logarithmic phase. The primary culture was initiated at 37 °C with 5% CO2 with medium refreshed at 5 day intervals. Once a confluent monolayer had formed, HCE cells were subcultured with 0.25% trypsin as described previously [In vitro culture of HCE cells was initiated as described previously . Donatedeviously . Briefly5 cell/ml, and the growth properties of the cells were measured as previously described [Passage 101 HCE cells were harvested and suspended in 20% FBS-DMEM/F12 medium to a density of 2.0×10escribed . BrieflyThe chromosome specimens of the passage 101 HCE cells at logarithmic phase were prepared as previously described . Briefly2. The medium was refreshed at 3-day intervals with the same BCS-containing medium, respectively, and the morphological changes of HCE cells were monitored daily under a Nikon E200 microscope .Morphology recovery of HCE cells at passage 101 was performed with 5%, 10%, 15% BCS-supplemented MEM, DMEM and DMEM/F12 medium (Invitrogen), respectively, as previously described . BrieflyTotal protein was extracted from passage 101 HCE cells, and 18 μl of protein extracts were directly applied to 7.5% sodium dodecylsulfate polyacrylamide gel. Gel samples were electrophoresed and electroblotted onto a 0.45 μm nitrocellulose membrane using an electroblotting system  according to the manufacturer’s instructions. After blocking with 10% BCS (Invitrogen) for 1 h at 37 °C, the blots were probed with mouse anti-human monoclonal antibodies of collagen type IV, von Willebrand factor (vWF), FLK-1, and keratin 12  overnight at 4 °C, respectively. The negative control blots were incubated with 0.05 M Tris-NaCl buffered saline (TBS) (pH7.2). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  was used as secondary antibodies. The blots were developed with kits of 3,30-diaminobenzidine tetrahydrochloride (DAB) in TBS .2O2 (Sigma-Aldrich), and blocked with 5% BCS (Invitrogen), the HCE cells cultured on coverslips were in turn incubated with rabbit anti-human NSE and HRP-conjugated goat anti-rabbit IgG, and DAB kit stained specimens were photographed with a Nikon E200 microscope. Omission of primary antibodies was used as negative controls.Immunocytochemistry of NSE expression of HCE cells at passage 101 was performed with rabbit anti-human NSE , HRP-conjugated AffiniPure goat anti-rabbit IgG  and DAB kits (Biosynthesis Biotechnology) as previously described  . BrieflyImmunofluorescence staining of cell-junction protein expression of HCE cells at passage 101 was performed with goat anti-human zonula occludens protein 1 (ZO-1) polyclonal antibody , mouse anti-human N-cadherin monoclonal antibody , mouse anti-human connexin 43 monoclonal antibody , mouse anti-human integrin αv/β5 monoclonal antibody , and fluorescent isothiocyanate (FITC)-conjugated rabbit anti-goat IgG antibody or rhodamine B isothiocyanate (RBITC)-conjugated goat anti-mouse IgG antibody or FITC-conjugated goat anti-mouse IgG antibody  as previously described , and visAQP1), Na+/K+-ATPase α1 polypeptide (ATP1A1), voltage-dependent anion channel 2 and 3 , chloride channel protein 2 and 3 , sodium bicarbonate cotransporter 4 (NBC1), and cystic fibrosis transmembrane conductance regulator (CFTR), was performed on the ABI Prism 7500 System . The primer sequences for these reactions were designed on the basis of the published human gene sequences (GAPDH) was used as an internal control. Quantification of the amplified product was done on a cycle-by-cycle basis via the acquisition of a fluorescent signal generated by binding of the fluorophore SybrGreen I  to double-stranded DNA. Each sample was assayed in duplicate with Taqman Universal PCR Master Mix . The cycling conditions were as follows: 10 min at 95 °C and 40 cycles of amplification for 15 s at 95 °C and 1 min at 60 °C. The quantification data were analyzed with the 7500 System Software (Applied Biosystems) according to the manufacturer’s instructions. Data shown are the mean±SD of three experiments.Real-time PCR was performed according to the methods described by Xiao et al. . Total Requences . The hou7 HCE cells at logarithmic phase was inoculated subcutaneously into one of the forehand oxters of each of 10 BalB/c nude mice respectively (n=10), while 10 BalB/c nude mice inoculated with 1.0×107 HeLa cells per mouse were used as positive controls. The tumorigenic status of the inoculated mice was monitored daily. After 60 days, the skin of the oxter of inoculated mice was surgically opened and tumorigenic status was examined.Tumorigenesis assay of the HCE cells at passage 101 was performed as previously described . About 16 cell/ml with 20% FBS-containing DMEM/F12 medium. Then 1 ml cell suspension was plated into 24 well culture plates coated with dAMs, and cultured at the same conditions as described above. Culture medium was refreshed entirely at 3 day intervals and the morphology and growth status were monitored daily. The morphology of cell sheets formed was examined with a Nikon Eclipse TS100 inverted microscope and a JSM2840 scanning electron microscope . The cell junction status was evaluated with 1% alizarin red staining and immunocytochemistry (the same method as described above). The continuous monolayer state of HCE cell was examined by freeze section and hematoxylin-eosin (HE) staining, and the attachment status of cell sheets and dAMs were examined with a H700 transmission electron microscope .Biocompatibility assay of HCE cells to dAM was performed as previously described . PassageData are expressed as mean±SD in triplicates or decuplicates and analyzed for statistical significance with ANOVA single factor.Numerous pure HCE cells were found to attach to the bottom of the wells when the corneal pieces were detached 36 h after initiation of attachment . And theChromosome analysis showed that HCE cells at passage 101 exhibited chromosomal aneuploidy with chromosome numbers ranged from 41 to 50, and the ratio of HCE cells with a chromosome number of 46 is about 47.8% , suggestAfter passage 101 HCE cells were cultured in 5%, 10% and 15% BCS-containing MEM, DMEM, and DMEM/F12 medium, respectively, for 2 passages, morphological changes of the cells occurred. The HCE cells cultured in 5% BCS-MEM medium underwent dramatic morphological changes from fibroblastic to polygonal on day 10 , implyinResults of marker protein expression are shown in Immunofluorescence staining pattern of cell-junction proteins are shown in AQP1, ATP1A1, VDAC2, VDAC3, ClC2, ClC3, NBC1, and CFTR  [±10.96 h To characterize corneal endothelial cells, various marker proteins such as NSE , collagen type IV (a marker protein of HCE cells), FLK1 , vWF , and keratin-12  have often been used ,16,23-26Since cell junctions are crucial for HCE cells to maintain intact endothelium for achieving stable corneal hydration status, determination of the expression of junction proteins was vital in HCE cell characterization . HCE cel+/K+-ATPase, VDACs, CLCs, NBC1 and CFTR, play crucial roles in maintaining corneal dehydration and transparency [+/K+-ATPase for maintaining corneal dehydration and transparency [+ and HCO3- [3- transport [AQP1, ATP1A1, VDAC2, VDAC3, CLC2, CLC3, NBC1, and CFTR, indicated that the established HCE cell line still had potencies to carry out normal functions of transmembrane transport, consistent with the results obtained from rabbit corneal endothelial cell lines [Membrane transport proteins of HCE cells, such as AQP1, Nasparency ,32-34. Asparency , and Na+sparency , VDACs fsparency ,38, CLCssparency ,40, NBC1nd HCO3- , and CFTransport ,43. In tll lines .Immortalized corneal endothelial cell lines by oncogene transfections may have latent potency of tumorigenicity and abnormal phenotypes, and cannot be used in studies of HCE cells and reconstruction of the ocular surface with a tissue-engineered cornea ,17. Tumo2, similar to that of a 10–11 years old child [Excellent biocompatibility between corneal cells and scaffold carriers is a vital precondition for in vitro reconstruction of tissue-engineered corneas ,44. Amonld child , which wld child ,27,46. Tld child ,27. The ld child .In conclusion, a continuous untransfected and non-tumorigenic HCE cell line, with normal protein expression and excellent biocompatibility to dAMs, has been established successfully in this study. And the cell line provides a powerful tool for studies of proliferation, differentiation and apoptosis of HCE cells. It could also be used for reconstruction of TE-HCE, providing a promising method for the treatment of diseases caused by corneal endothelial disorders. Studies on TE-HCE reconstruction and its animal transplantation are currently underway in our laboratory.


Embryonic neuroepithelia and adult subventricular zone (SVZ) stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP) specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes. Expression of the intermediate filament protein nestin has been used as a marker for neural stem and progenitor cells in the ventricular and subventricular zones –3. NestiTransgenic reporter mice, even when generated identically, can have widely divergent expression of the transgene . Indeed,Nestin, an intermediate filament protein, is expressed by neural stem and progenitor cells , 17, 18.At P0, many eGFP+ cells were found in the caudatopallial angle and lateral migratory stream . These cAnother report of nestin-eGFP mice suggested that eGFP fluorescence intensity was either dim or bright . In our Endogenous nestin has been reported in all three of the major neural SVZ cells  . We firsin vitro from proliferative neuroepithelia can exhibit self-renewal and multipotency, the two cardinal features of stem cells. We examined the ability of eGFP+ cells to make neurospheres. Similar to other nestin-GFP lines [We next studied the mitotic potential of eGFP+ cells by administering BrdU for two weeks in four-week-old mice. eGFP+ cells in the postnatal SVZ and RMS  were labFP lines , 9, 12, eGFP was also expressed by scattered cells throughout the brain parenchyma at all ages examined, and were especially dense in the piriform cortex . Many ofeGFP was expressed in oligodendrocyte progenitors; in addition, some eGFP+ cells in the SVZ were GFAP+. Therefore, we asked whether eGFP was expressed in astrocytes outside of the SVZ, in the parenchyma. We labeled with anti-GFAP antibodies and did not find any double labeled cells .N = 4 mice, 89 cells examined), at 2 days , 5 days , and 10 days  after injury. We did not find a single eGFP+ cell in these regions that was colabeled with GFAP  and 1 μL of 10 mg/mL Proteinase K and incubated at 55°C, overnight, and 1-2μL of the processed, lysed tail DNA was used for each PCR reaction.Standard molecular biology techniques were used to generate the transgenic constructs. Briefly, the second intron from the rat nestin gene , 41 was Were performed as described previously –44. AdulBrdu (200 mg/kg) was injected intraperitoneally in 4-week-old eGFP+ mice every 12 hrs for two weeks.N = 4, P0 N = 3, P14 N = 4, P28 N = 1, P30 N = 3, P63 N = 1, P84 N = 4, and P224 N = 1. Mice were anesthetized at 4°C with wet ice between E162-P14. Ages P28–P224 were anesthetized with sodium pentobarbital and perfused with 4% paraformaldehyde in 0.1 M sodium phosphate buffered saline (PBS), pH 7.6. Thirty micron free-floating coronal sections were cut on a sliding microtome and stored in cryoprotectant at −20°C. All animal procedures were in accordance with NIH animal care guidelines and were approved by the Children's Memorial Hospital IACUC committee.Male and female eGFP+ mice ranging between ages E16-P224 were used: E16 2 flasks coated with Poly-HEME  in Neurobasal (Invitrogen) supplemented with the growth factors fibroblast growth factor (FGF-2)  and epidermal growth factor (EGF) , as well as B-27 Supplement (Gibco), N-2 Supplement (Gibco), L-glutamine , and Penicillin/Streptomycin . Neurospheres were dissociated and passaged 9 days after seeding. Cultures were kept for at least 4 passages.The lateral wall of the lateral ventricle, including the SVZ, was dissected from coronal slices of P5 mouse brains, dissociated with 200 mL Papain , and plated at a density of 3,000 cells/mL in 25 cmThe following antibodies were used: anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase , antipan Dlx    for 10 min before the final rinses. Negative controls omitting the primary antibodies were carried out in all experiments for all the secondary antibodies used. Sections were analyzed with Leica epifluorescent microscopes controled by Openlab software  or an Olympus Confocal microscope controlled by Fluoview software . All GFP+ cells were imaged with 40x or 63x objectives and by shifting between the GFP, Cy3, and DAPI filter cubes. Images generated with these systems were assembled in Adobe Photoshop.N = 1 mouse, P45). The brain was vibratome sectioned at 50 μm and washed in PBS, 6x, 10 min. Aldehydes were blocked by 30 min incubation in 50 mM glycine in PBS. Sections were washed in PBS 3 × 10 min, incubated in 5% normal goat serum, 0.1% triton-x 100 in PBS (PBS+), and rabbit anti-GFP 1 : 1000 added for overnight incubation at 4°C. Sections were washed in PBS, 3x, 10 min, incubated for 60 min, RT, in antirabbit conjugated 1.4 nm gold  in PBS+. Sections were washed in PBS, 3x, 10 min, PB, 3x, 10 min, DH2O, 3x, 10 min, then incubated in Gold Enhance solution (Nanoprobes) for 5 min, washed in DH2O, 3x, 5 min, PB 3x, 5 min, fixed in 1% gluteraldehyde for 30 min, washed in PBS, fixed in 1% osmium tetroxide in PBS for 10 min, washed in PBS, dehydrated in EtOH, infiltrated in 1 : 1 propylene oxide : epon for 1 hr at RT, 1 : 2 PO : epon for 2 hrs, epon for 2 hrs, and embedded in epon overnight at 60°C. Stained ultrathin sections in 2% aqueous uranyl acetate for 10 min, washed in DH2O, and viewed on a JEOL electron microscope equipped with a digital camera.Mice were perfused with 2% paraformaldehyde and 0.1% glutaraldehyde in PBS and left in same fixative overnight at 4°C (


Our data support the use of TCL1A in the panel of diagnostic markers used in the differential diagnosis of splenic low-grade B-cell lymphoma; a possible prognostic value, however, needs a larger series to be established.Splenic marginal zone lymphoma (SMZL) is a low-grade lymphoma showing a rather nonspecific immunophenotype. Gene expression profiling studies suggested that TCL1A could be a marker of SMZL, but reported data are conflicting. We evaluated TCL1A expression in a series of spleen and bone marrow samples involved by SMZL and correlated the findings with other immunophenotypical, morphological, and clinical data. In addition, we evaluated the expression of TCL1A in a series of spleens and lymph nodes involved by lymphomas that might mimic SMZL , 39 follicular lymphomas (FL), 30 B-cell chronic lymphocytic leukemias (B-CLL), 31 mantle cell lymphomas (MCL), 1 lymphoplasmacytic lymphoma) and 15 bone marrow specimens involving hairy cell leukemia (HCL). TCL1A staining was negative in 24/31 cases of SMZL (77 %); 27/31 MCL and all B-CLL were positive for TCL1A; 32/34 cases of nodal FL (96 %) and all five splenic FL were positive for TCL1A, although at a lower intensity. Eight of 13 NMZL were positive for TCL1A, often showing a heterogeneous staining pattern. All HCL samples were strongly positive for TCL1A. No correlation was found between the pattern of splenic infiltration, TCL1A expression, and the clinical course. TCL1A-positive SMZL showed a higher rate of DBA44 staining compared to the negative ones, and this difference was statistically significant (Fisher test, single-tailed,  Splenic B-cell marginal zone lymphoma is a rare entity, accounting for less than 2 % of all lymphoid malignancies; it shows an indolent clinical course, although 10 % of cases progress to diffuse large B cell lymphoma [A commercially available specific marker of SMZL is lacking, and the immunophenotypical profile of SMZL is currently mainly defined on the basis on the lack of expression of markers associated with its mimickers , including CD5, CD10, CD23, cyclin D1, CD25, CD11c, and Annexin A1 to name just a few. However, both SMZL and its mimickers can occasionally show an aberrant phenotype ), making a final diagnostic decision more difficult. The detection of the deletion of 7q might help, because it is rare in other lymphoma subtypes, while it is present in about 30–40 % of SMZL .TCL1A (T-cell leukemia/lymphoma 1A) is a proto-oncogene member of a multigene family that includes TCL1B and MTCP1 [TCL1A maps to the 14q32.1 locus and has been shown to enhance the activity of the Akt kinase, which plays a central role in regulating cellular survival [TCL1A acts like a classic oncogene through constitutive activation [Although not widely used, TCL1A is a helpful marker in lymphoma characterization . TCL1A , and disease-free survival (DFS).Nine cases of non-Hodgkin lymphoma involving the spleen were used as controls, including five follicular lymphomas (FL), one mantle cell lymphoma (MCL), one small B-cell lymphoma/chronic lymphocytic leukemia (B-CLL/SLL), one hairy cell leukemia (HCL), and one lymphoplasmacytic lymphoma (LPL). Five normal spleen specimens (removed after traumatic injury) were also included in the study. We also added a series of lymph node cases involving lymphomas that might mimic SMZL  and 15 bone marrow specimens involving hairy cell leukemia, which might be a differential diagnostic problem when only minimal involvement is present.Cases were classified morphologically as described by Isaacson et al. . Spleen Three tissue microarrays were built using a manual arrayer (Beecher Instruments). After morphological evaluation and marking of proper areas in H&E-stained slides, three 2-mm cores were taken from each block. All slides were scanned using the D-SIGHT device .Immunohistochemical staining was performed using an autostainer (Leica Bond Max) using the following antibodies: CD5, CD20, Ki67, cyclin D1, CD123, TCL1, T-bet, DBA44, BCL2, BCL6, CD23, ANXA1, MNDA, and GCET1 (Table Fisher exact test (one-sided and two-sided) was used for statistical analysis.Clinical data were available for 24 cases. Mean age was 62 years (range 48–79). All cases presented with splenomegaly  and all underwent splenectomy for diagnostic and therapeutic purposes. Fourteen of 24 cases (58 %) presented with liver enlargement and 17/24 with lymphadenopathy. In all patients with an available bone marrow trephine biopsy, an involvement was demonstrated.Other relevant findings were lymphocytosis , anemia , thrombocytopenia , presence of serum M component , and autoimmune phenomena ; 2/24 (8 %) patients resulted HCV positive and other two (8 %) HBV positive. One third of patients  presented an IPI score of 3–4, while 15/24 (62 %) an IPI score of 1–2; data were not available for one case. Seven of 24 (29 %) presented LDH values above normal range; 3/24 (12 %) presented an albumin concentration below 35 g/L. Sixteen of 24 patients (66 %) had normal values of albumin, LDH, and hemoglobin; 3/24 patients (12 %) had one of these three values outside the normal range, 3/24 patients (12 %) had two abnormal values, and 2/24 (8 %) had all three values outside the reference range.As a first line of therapy, 9/24 (37 %) patients were treated with chemotherapy, consisting of alkylating agents with or without rituximab besides splenectomy; the remaining 15 patients underwent splenectomy alone. Of these, 11 underwent chemotherapy as second-line therapy. The mean follow-up period was 69 months (range 6–233). During the follow-up period, 5/24 patients (20 %) died because of the lymphoma, after a mean period of 80 months (range 49–110); 9/24 (37 %) patients presented signs of disease progression. At the time of the last clinical observation, 7/24 patients (29 %) were free from disease after chemotherapy/splenectomy.In the spleen, a nodular pattern of infiltration was present in 8/31 cases 26 %), a nodular/diffuse pattern in 15/31 cases (48 %), and a primarily diffuse pattern in 8/31 cases (26 %). In bone marrow biopsies, we found a mixed nodular/interstitial/sinusoidal pattern in 9/25 cases (36 %), nodular/sinusoidal in 5/25 (20 %), interstitial/sinusoidal in 4/25 (16 %), nodular/interstitial in 1/25 (4 %), purely sinusoidal in 3/25 (12 %), and purely interstitial in 3/25 (12 %)  but became significant when using a single-tailed test (p = 0.0397). T-bet resulted positive in 7/31 cases (22 %), while ANXA1, cyclin D1, and CD123 were invariably negative. In one case, the neoplastic cells were CD5 positive, but negative for CD23, cyclin D1, and TCL1A; thus, a splenic localization of CLL/SLL or MCL was reasonably excluded. TCL1A-positive cases showed no apparent peculiarities when compared to the negative ones; the staining was present usually only in part of the cells (36–75 % in six of seven cases) and with variable intensity  cases showed a variable degree of staining for DBA44, although only 5/30 (17 %) in more than 35 % of cells. Five of the six TCL1A-postive cases where also DBA44 positive (83 %), while 8/24 TCL1A-negative cases showed expression of DBA44 (33 %). This difference was not statistically significant using a two-sided Fisher exact test  of NMZL showed a positive but heterogeneous staining with a “mosaic-like” pattern Fig. . Twenty-No correlation was found between the type of splenic infiltration and the clinical course, including OS and DFS. We did not find any statistically significant correlation between TCL1A expression and the primarily diffuse pattern seen in 7/31 cases, nor with disease progression. In fact, we did not find any difference in terms of TCL1A expression among cases with a diffuse pattern of splenic infiltration compared to those with a nodular or nodular and diffuse pattern.Due to the lack of specific markers, splenic marginal zone B-cell lymphoma is still a diagnosis of exclusion. The use of a large number of markers, even negative, allows exclusion of morphologically similar entities and thus improves diagnostic reliability. The problem is more pertinent when analyzing bone marrow specimens, since the diagnostic criteria are less reliable, and morphological evaluation is sometimes of limited help. Since only a part of the patients with SMZL undergo splenectomy before a bone marrow biopsy is taken, these specimens are often the first (and frequently the only) samples available for evaluation.In this paper, we analyzed the expression of TCL1A in SMZL, a marker that is lost in marginal zone differentiation of normal B cells , the mRNOur series of cases showed an indolent course and good prognosis, similar to previously published studies. Only 5/24 patients (21 %) for whom clinical data were available died because of lymphoma, after a median interval of 80 months (range 49–110). Nine of 24 patients (37 %) underwent disease progression, three of whom developed transformation to diffuse large B-cell lymphoma.p = 0.0606). Since we wanted to test the positive association between TCL1A and DBA44, a one-tailed test might be appropriate which turned out to be significant (p = 0.0397). The small number of cases did not allow a more powerful statistical approach, but the high prevalence of double-positive cases is intriguing and warrants further investigation of the hypothesis that DBA44-positive cases might be different from the negative ones, irrespective of the growth pattern.Morphologically, a purely diffuse pattern was present in 8/31 26 %) of spleen samples of SMZL, while the majority showed a classical nodular pattern with or without red pulp involvement. Three of the purely diffuse cases were also positive for DBA44 and, based on the 2008 WHO criteria, would fit in the provisional category called “splenic B-cell lymphoma, unclassifiable,” which includes a subcategory called splenic diffuse red pulp lymphoma. DBA44 was not restricted to the purely diffuse subtypes, however, since also ten other cases with mixed or nodular patterns showed a variable degree of expression  of cases of SMZL showed positive staining for TCL1A, among cases of lymphomas that might mimic SMZL, 8/13 (61 %) of NMZL showed a mosaic-like, heterogeneously positive staining, while all nodal B-CLL/SLL (29/29) and 27/30 (90 %) nodal MCL showed strong and diffuse positivity for TCL1A (the remaining three MCL cases showed a weak or heterogeneous staining). Moreover, 32/34 nodal cases of FL (94 %) and all HCL bone marrow trephine biopsy (15/15) stained positive for TCL1A. In the spleen, five FL, one MCL, one B-CLL/SLL, one HCL, and one LPL also showed expression of TCL1A.As previously said, among the five cases that were positive for TCL1A on spleen specimens and had paired bone marrow biopsies, one case was negative in the bone marrow biopsy. This asymmetry might be due to the interaction between neoplastic cells with the bone marrow microenvironment or simply to a lower technical performance on de-calcified samples .IGHV genes. In splenic marginal zone B-cell lymphomas, this latter correlation has never been directly proven, but indirect evidence comes from studies that demonstrated that the presence of wild-type IGHV genes correlates with the presence of deletion of 7q31 [In a study conducted in 2006 by Herling et al. , the exp of 7q31  and that of 7q31 . Furtherp < 0.05) is present if the three abnormal parameters are taken together and correlated with TCL1A-positive staining. TCL1A expression might therefore be a predictor of shorter survival, but the low number of TCL1A-positive cases does not allow a powerful statistical analysis.In a multicenter study conducted by the Intergruppo Italiano Linfomi, Arcaini et al.  suggesteRecently, Kanellis et al.  evaluate


Objective. To investigate the expression and clinical significance of trans-membrane MerTK (mMer) on circulating CD14+ monocytes/macrophages and soluble MerTK (sMer) levels in plasma in systemic lupus erythematosus (SLE). Method. 108 SLE patients and 42 healthy controls were recruited in this study. The expression of mMer on the surfaces of CD14+ monocytes/macrophages was evaluated by flow cytometry (FCM). The sMer levels were measured by ELISA. Real-time quantitative PCR was applied to evaluate the mRNA levels of MerTK and ADAM17. Results. Both mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma significantly increased in SLE patients compared to healthy subjects. The frequency of anti-inflammatory MerTK expressing CD14+CD16+ monocytes decreased in SLE. mMer expression was positively correlated with CD163 expression on CD14+ cells. Both the mMer expression on CD14+ monocytes/macrophages and sMer levels in plasma were positively correlated with SLEDAI. Furthermore, more elevated mMer and sMer levels were found in patients with higher SLEDAI, presence of anti-SSA, anti-Sm autoantibodies, and lupus nephritis. Conclusion. Both mMer and sMer levels significantly increased in SLE and positively correlated with disease activity and severity. The upregulation of MerTK expression may serve as a biomarker of the disease activity and severity of SLE. Mer tyrosine kinase (MerTK) is an integral membrane protein that is preferentially expressed in hematopoietic lineages such as monocytes/macrophages, dendritic cells (DCs), and natural killer (NK) cells , 2, whicSystemic Lupus Erythematosus (SLE) is an autoimmune disease with multiorgan damage characterized by defective phagocytosis of ACs, release of inflammatory cytokines, and aberrant activation of autoreactive T and B cells, with subsequent production of pathogenic autoantibodies against cell nuclear components and resultant end-organ injury . ImpaireIn this study, we determined the expression levels of mMer on circulating CD14+ monocytes/macrophages and sMer levels in plasma from patients with SLE and analyzed the clinical significance of both mMer and sMer in SLE. Our study showed that both of the mMer and sMer levels significantly increased in SLE and positively correlated with disease activity and severity. Meanwhile, we investigated the different mRNA expression of MerTK and ADAM17 between SLE patients and healthy controls to further demonstrate possible reason for increased shedding of MerTK in SLE. Furthermore, we determined the frequency and MerTK expression pattern of M2c-like CD14+ (bright) CD16+, CD163+ monocyte/macrophage population in comparison to healthy subjects .B samples were obtained from 108 consecutive patients with SLE  enrolled at the ward of the department of rheumatology and immunology, Peking University People's Hospital from July 2012 to February 2013. All patients fulfilled at least 4 of the 2010 American College of Rheumatology (ACR) revised criteria for SLE . 42 heal9/L and 100 × 109/L were regarded as leukocytopenia and thrombocytopenia, respectively. Proteinuria was defined as 24 h proteinuria excretion more than or equal to 0.5 g. Anti-dsDNA Ab, ACL, AnuA, C3, C4, IgG, IgM, and IgA were tested by ELISA, with normal ranges of 0–100 IU/mL, 0–12 RU/mL, 0–20 RU/mL, 0.88–2.01 G/mL, 0.16–0.47 G/mL, 6.94–16.18 G/mL, 0.6–2.63 G/mL, and 0.68–3.78 G/mL. ANA, SSA, and Sm were tested by indirect immunofluorescence assay. Positive auto-antibodies of anti-dsDNA Ab, ACL, and AnuA were defined as values more than 100 IU/mL, 12 RU/mL, and 20 RU/mL, respectively. Decreased C3 and C4 were defined as values less than 0.88 G/mL and 0.16 G/mL. CRP was examined by immunonephelometry method. Values more than or equal to 7.9 mg/L were considered positive.General and Laboratory data from the medical records of these patients include age, gender, disease duration, clinical symptoms, blood cell counts , routine chemistry, urinalysis, 24 h proteinuria excretion, lupus associated anto-antibodies , immunoglobulins , Complement component 3 (C3), Complement component 4 (C4), and C-reactive protein (CRP). White cell and planet counting less than 4 × 10Disease activity was calculated by using the SLE disease activity index (SLEDAI) . Clinicaμg of total RNA by using random oligonucleotides as primers and a RevertAid First Strand cDNA Synthesis Kit (Thermo Science Corporation). Gene expression was assessed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MerTK, and ADAM17 using the following primers: GAPDH , MerTK , and ADAM17 . PCR amplification was performed by using SYBR Green assay with the following thermal step: initial denaturation at 94°C for 3 minutes (min), followed by 40 cycles of denaturation at 94°C for 30 seconds (s), annealing at 58°C for 30 s, and extension at 72°C for 30 s. The 7300 Detection System (AB Applied Bio-systems) was used to run quantitative real-time PCR of the samples according to the manufacturer's instructions. Reactions were run in triplicate and generated products were analyzed with the SDS software. mRNA levels were expressed as threshold cycle (CT). For relative quantification, the expression target genes were normalized by expression of GAPDH gene. The data was evaluated as 2−ΔΔCt values. Results were expressed as relative quantity to the control as normalization ratio where all the other samples were compared in terms of their fold difference to the control.Human PBMCs were obtained from the venous blood of 35 SLE patients and 26 healthy controls by Histopaque density gradient centrifugation using human peripheral leukocyte isolation liquid . Magnetic separation kit (Miltenyi) was used for human CD14(+) monocytes/macrophages enrichment from PBMCs of another 8 SLE patients and 5 healthy controls by positive selection according to the manufactures' instructions. Purity of CD14(+) cells was >95%. RNA in trizol reagent was extracted from PBMC and CD14(+) cells mentioned above with RNA simple total RNA kit , and cDNA was synthesized from 1 μL volume of blood was used for each subject. Containing serum components were removed by washing the cells three times in an isotonic phosphate buffer  by centrifugation at 1600 rmp for 5 min. 50 μL of packed cells was then transferred to 5 mL tubes for staining with monoclonal antibodies. Before staining with antibodies, cells to be used were firstly Fc-blocked by treatment with 5 μL Fc receptor blocking solution  per 50 μL of suspension for 15 min at room temperature. The following antibodies were added to the tube: anti-human CD14 , anti-human CD16 , anti-human CD163 , and anti-human MerTK . Corresponding negative isotype and fluorochrome-matched controls were used in a separate tube. After following the incubation protocols as recommended by the respective manufacturers, 2 mL 1% flow cytometry solution  for lysing red blood cells was added to the whole cells for 10 min in the dark at room temperature. Then, the cells were washed twice in 4 mL of sample PBS buffer. The cells were resuspended in 400 μL of PBS buffer for final flow cytometric analysis. The stained cells were processed in flow cytometry (BD FACS AriaTM II). A forward scatter-side scatter plot was used to gate lymphocytes, monocytes/macrophages, and granulocytes (Venous blood samples (4 mL) were obtained from all subjects in purple tubes containing ethylenediamine-tetraacetic acid (EDTA) as the anticoagulant. 100 ulocytes . The perulocytes . Likewisulocytes . The res2HPO4, 1.5 mM KH2PO4, PH 7.2–7.4, 0.2 μm filtered.). The plates were washed three times with 0.05% Tween-20 in PBS during each step. An eight-point standard curve was made by 2-fold serial dilution of recombinant proteins and blank controls were reagent diluent alone. No plasma dilution was performed for sMer detection. The antigen was detected by a biotinylated goat anti-human Mer antibody  and streptavidin conjugated to horseradish peroxidase (HRP) . The tetramethylbenzidine (TMB)  was added as the substrate solution and the color reaction was stopped by the addition of 50 μL 2 N sulphuric acid. The absorbance was read at 450 nm with a correction wavelength set at 570 nm using a microplate reader . sMer concentrations were calculated using ELISA calc. regression computer software by creating a standard curve through reducing the blank data to generate a four-parameter logistic (4-PL) curve-fit prepared from 2-fold serial dilutions of recombinant MerTK.Plasma sMer was determined by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA). Blood examples were collected into EDTA tubes and centrifuged at 1600 rmp for 10 min. The plasma was subpacked and aliquots were stored at −80°C until assayed. The DuoSet development system for sMer (DY6488) was purchased from R&D Corporation . 96-well plates were coated overnight with MerTK capture antibody. The plates were blocked with reagent diluent containing 1% BSA in phosphate buffered saline  version 16.0 was used to analyze the data. Experimental data were expressed as the mean ± standard deviation and statistical significance between two groups was assessed with the Student's paired P = 0.1; gender: X2 = 1.234, P = 0.124). The SLE patients had mean disease duration of 68.16 months ranging from 1 to 420 and the mean SLEDAI score of these patients was 9.44 ranging from 0 to 48.Demographic and clinical characteristics of SLE patients and healthy controls are shown in n = 35, 8.69 ± 2.28 versus n = 26, 9.16 ± 1.6, P = 0.876; n = 8, 0.20 ± 0.02 versus n = 5, 0.23 ± 0.04, P = 0.497, resp.). The ADAM17 mRNA level in PBMC was significantly lower in SLE patients than that in healthy controls . In CD14+ monocytes/macrophages, although the ADAM mRNA levels tended to decrease in SLE patients, there was no significant difference between the patients and the controls , which implicated that ADAM17 might play a role in promoting Mer shedding and sMer production.MerTK and ADAM17 mRNA expression were detected in both SLE patients and healthy controls. As showed in Figures  Figures . There wn = 42, 27.15 ± 2.88 versus n = 25, 8.84 ± 1.35, P < 0.001) as presented in The mMer levels on cell surfaces of CD14+ monocytes/macrophages were significantly increased in SLE patients than in healthy controls   (⩾27.8 was significantly increased (P = 0.008) than that in group with decreased CD163 expression defined as <27.8 in healthy controls (24.11 ± 4.89 versus 8.86 ± 1.69)  . Previou< 0.001) . We diviP < 0.001). However, significantly elevated mMer expression on this subset in SLE patients was also detected   .Our data showed that the elevation of mMer on CD14+ monocyte/macrophages was accompanied by the increased expression of CD163, which defined the CD14+CD163+ subset as the main CD14+ monocyte/macrophage population with elevated mMer expression, and mMer expression in the immune-regulatory CD14+CD16+ subset also increased in SLE.n = 108, 2170.30 ± 160.87 pg/mL) was also significantly higher (P < 0.001) than that in healthy controls    . There w = 0.19) .r = 0.343, P = 0.026)  and significant correlation was detected between sMer and 24 hours proteinuria excretion   . The pla= 0.002) .P < 0.001) and sMer levels in plasma . Plasma sMer levels were significantly elevated (P < 0.001) in patients with proteinuria (2582.3 ± 225.12 pg/mL) than those without protein excretion (1441.5 ± 139.49 pg/mL). Patients with presence of SSA, Sm, or lupus nephritis showed both higher mMer and sMer levels than those without SSA, Sm, or lupus nephritis   .Deregulation of innate immunity and clearance of apoptotic cells have been implicated in the pathogenesis of SLE , 21. In Membrane MerTK was reported to be shed into soluble forms through ADAM-17 dependent cleavage and circulated in plasma . AlthougIt was reported that sMer levels were correlated with disease activity of SLE , 34. ConRecent studies demonstrated that induction of MerTK expression enhanced phagocytosis of apoptotic debris and anti-inflammatory activity of the CD14+CD16+ M2c-like subset of macrophages , 35. In In summary, we demonstrated that both the mMer level on circulating CD14+ monocyte/macrophage and sMer level in plasma significantly increased in SLE, and they positively correlated with disease activity and severity. In SLE, the circulating M2c-liked CD14+CD16+ monocyte/macrophage subset, which was reported to play an anti-inflammatory role, showed increased mMer expression but its frequency significantly decreased. The upregulation of MerTK expression may serve as a biomarker of the disease activity and severity of SLE.ADAM17 has been proved to play a vital role in shedding of MerTK in animal model (Reference 6). To further study the role of ADAM17 in mMer shedding and sMer production, our data demonstrated the correlation between ADAM17 mRNA levels in PBMCs and sMer levels in plasma (supplementary figure 1). In the supplementary figure 2, we have included the representative flow cytometry diagram which showed that higher CD163 expression on CD14+ Mo had higher Mer expression.Click here for additional data file.


These results suggest that sifuvirtide gel is an effective, safe and stable product, and should be further tested as a vaginal or rectal microbicide in pre-clinical model or clinical trial for preventing HIV sexual transmission.Sifuvirtide is a proven effective HIV-1 entry inhibitor and its safety profile has been established for systemic administration. The present study evaluated the potential of sifuvirtide formulated in a universal gel for topical use as a microbicide candidate for preventing sexual transmission of HIV. Our data showed that sifuvirtide formulated in HEC gel is effective against HIV-1 B, C subtypes, CRF07_BC and CRF01_AE, the latter two recombinants represents the most prevalent strains in China. In addition, we demonstrated that sifuvirtide in gel is stable for at least 8 weeks even at 40°C, and did not cause the disruption of integrity of mucosal epithelial surface, or the up-regulation of inflammatory cytokines both  Recent report from Joint United Nations Programme on HIV/AIDS (UNAIDS) showed that the global HIV-1 new infections have declined by 19% in year 2010 compared with that in year 2009 HIV-1 infection could be dissected into multiple consecutive steps, including binding, fusion, entry, reverse-transcription, integration, transcription, translation and cleavage, assemble and budding; Candidate drugs targeting procedures other than reverse transcription are also likely to be effective if used as active components in microbicide candidate. Similar to highly active antiretroviral therapy (HAART), the most effective strategy for microbicide development may need to combine several antiretroviral drugs targeting at different steps during HIV-1 life cycle. Since HIV-1 viral entry is considered as an important target in combating HIV/AIDS infections, several entry inhibitors specifically blocking virus-cell attachment and fusion events have been evaluated preclinically as potential microbicides, including CCR5 antagonists , lectins , and monoclonal antibodies (mAbs) that inhibit HIV-1 binding or entry through binding to viral envelope glycoprotein gp120, gp41, cellular receptor CD4 or coreceptor CCR5 in vitro, and SFT gel displayed good biosafety and stability in both in vitro and in vivo evaluation models. Consequently, these findings suggest the potentials of SFT gel formulation for intravaginal or intrarectal application as a microbicide to prevent the sexual transmission of HIV-1.Sifuvirtide (SFT) is a new HIV-1 fusion inhibitor whose design was based on the three-dimensional structure of the HIV-1 gp41 fusogenic core conformation Nonoxynol-9 (N-9)  and cellulose sulfate (CS)  were purchased from ScienceLab.com, Inc. ; κ-Carrageenan  was purchased from Sigma-Aldrich  and TFV  was purchased from Molekula Limited . Sifuvirtide (SFT) was provided by FusoGen Pharmaceuticals, Inc. .in vitro experiments on cell lines, SFT was dissolved in sterile PBS (pH = 7.4) at indicated concentrations, to which hydroxyethyl cellulose (HEC) was added , followed by continuously stirring for 45 min to allow all solutes to dissolve. For in vivo application in mouse vagina, SFT, TFV, and N-9 were first dissolved in PBS (pH = 4.5) at indicated concentration, to which HEC was slowly added to reach the final HEC concentration of 1.5% while the solution was rapidly stirred until a translucent gel was formed in vitro and in vivo tests, respectively, based on an empirical rule in vitro should be diluted 100-fold from the concentration used for in vivo test to facilitate the even spread of the product over the cell culture and to avoid interference with the cell growth in culture. . CS and κ-Carrageenan were dissolved at indicated concentration in pH 4.5 PBS without HEC to form gel formulations automatically. All gels were allowed to equilibrate overnight at room temperature and were retested for pH before intravaginal application as described below.For 2.A human colorectal epithelial cell line, Caco-2, was kindly provided by ATCC (USA) and was cultured in Dulbecco's Modified Eagle Medium (DMEM); TZM-bl cells and mouse leukemic monocyte macrophage cell line RAW 264.7 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and cultured in DMEM and RPMI 1640, respectively. All cell cultures were supplemented with 10% fetal bovine serum (FBS), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C/5% CO4 TZM-bl cells per well were plated into a 96-well plate in DMEM containing 10% fetal bovine and penicillin-streptomycin. After culture at 37°C for one day, pseudoviruses (100 TCID50 per well) were mixed with SFT or TFV in PBS solution or HEC gel formulation at the indicated concentration, and then the mixtures were added to the cells, followed by an incubation at 37°C with 5% CO2 for 48 h. The cells were then lysed in the presence of Bright-Glo (Promega), and relative luminescence was recorded by using a Victor 3 luminometer (PerkinElmer). The IC50 was then calculated.In the present study, we tested antiviral activity of SFT in PBS or gel against viruses pseudotyped with Env of HIV-1 strains that are predominantly circulating in China, HIV-1 subtype B and subtype C. TFV, an NRTI that was proven in clinical trial to reduce the risk of women acquiring HIV-1 infection by 39% 90 value against HIV-1 subtype C) for testing their inhibitory activity on HIV-1 pseudovirus subtype C infection, as described above. In addition, the stability of SFT in the gel was further analyzed by high-performance liquid chromatography (HPLC) on samples stored at 40°C for up to 8 weeks. Their viscosity and pH profiles were also determined as described below. The sample without incubation at 40°C (0 week) was used as a control.An accelerated stability study of SFT in microbicide gel was conducted as previously described sample of week8/PAsample of week0×100”.HPLC analysis of SFT peptide in gel were conducted on an Agilent 1100 LC system (Agilent Technologies) with a reverse-phase Ultra Aqueous C18 analytical column  and a guard column  which were purchased from Agilent Corporation. Samples were diluted in 10% acetonitrile (ACN), 0.1% formicacid (FA). The LC elution conditions were set as follows : mobile phase A, 0.1% FA in water; mobile phase B, 0.1% FA in ACN; time program, 0 min, 95% A/5% B; 1.0 min, 95% A/5% B; 10 min, 40% A/60% B; 12 min, 5% A/95% B; 15 min, 5% A/95% B; 16 min, 95% A/5% B; 25 min, 95% A/5% B. The flow rate was 200 µl/min at ambient temperature. Column oven temperature was 25°C. The detector was monitored with UV detection at 215 nm. Peak area (PA) was calculated by Chemstation software (Agilent Technologies) and recovery of SFT was calculated as “PAThe viscosity of the gel formulations was measured by using a NDJ-5S Rotational Viscometer  with rotor #4 (60 rpm) at 25°C±0.1°C. The pH of the gels was determined at room temperature by using a 423 microprobe electrode .4 ml−1 in 96-well flat-bottom microtiter plates with different concentrations  of SFT in 0.015% HEC gel or in PBS solution. After an incubation for 12 h at 37°C in a humidified 5% CO2, 50 µl of RAW264.7 cells in medium were sampled and stored at −80°C for further cytokine quantification assay as described below. After continuous incubation for 48 h, 10 µl of MTT (5 mg/ml) were added to the plate and incubated for 4 h at 37°C. Afterwards, the medium was removed, and the formazan, a product generated by the activity of dehydrogenases in cells, was dissolved in acidified isopropanol (0.4 N HCl). The amount of MTT formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 490 nm by using a Bio Assay reader . The cell viability was calculated by the following equation: (OD treatment/OD control)×100. Experiments were carried out in triplicate.The effect of SFT gel or SFT PBS solution on the viability of Caco-2 cells or RAW264.7 cells was assessed by monitoring MTT metabolism using a colorimetric assay of cell survival which was carried out by the method of Denizot and Lang in vivo safety study, mice randomized into eight groups (5 mice/group) were intravaginally treated with 40 µl of 1.5% HEC gel containing 0.03 mM SFT, 0.3 mM SFT, 3 mM SFT, 1% N-9, 1% TFV, 6% CS, 3% carrageenan, or PBS (vehicle control), respectively. The corresponding gel formulations were then delivered intravaginally every 12 h (twice daily) for three consecutive days. Twelve hours after the last treatment, cervicovaginal lavages (CVLs) were collected by washing the cervicovagina with 200 µl of sterile saline. The collected CVLs were treated with protease inhibitor  and centrifuged at 200×g for 10 minutes at 4°C. Supernatants were then collected and stored at −80°C for subsequent cytokine quantification assay. Finally, the mice were sacrificed, and the vaginal tissues were collected for preparation of mononuclear cells (MNCs) or for histological examination as described below.All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Public Health Clinical Center and were performed in accordance with relevant guidelines and regulations. Six- to eight-week-old pathogen-free outbred BALB/c female mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. . All mice were hormonally synchronized with 2 mg of medroxyprogesterone acetate  5 days prior to the treatment with candidate microbicides Formalin-fixed excised vaginal tissues were embedded within paraffin and transversely sectioned with a microtome. The slides were stained with hematoxylin-eosin and subjected to a blind evaluation for epithelial cell disruption and inflammatory responses.Cytokines from RAW264.7 cell culture supernatants or cervicovaginal lavage fluid were determined by using a mouse cytokine CBA kit  and analyzed on a FACSAria flow cytometer (BD Biosciences). Tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-17A and IL-10 were included in the cytokine panel. Standard curves were generated for each cytokine. Prior to examination, the boundary was set from 0–5000 pg/ml, and the lower detection limit for different analytes ranged from 0.03–16.80 pg/ml, as described in manufacturer's instructions.t-test using GraphPad Prism, version 5.0 . Values of p<0.05 were considered statistically significant.Data are presented as the mean ± standard deviation (SD). Statistical significance between different groups was calculated by the Student's 50 ranged from 2.68 to 47.78 nM 50 values of SFT in PBS against CRF07_BC, CRF01_AE, subtype B and subtype C were 13.3, 30.9, 5.9 and 25.6 nM, respectively, while those for SFT in 0.015% HEC gel were 9.8, 22.2, 3.4 and 12.4 nM, respectively  or clinical evaluations. Intriguingly, TNF-α was observed to be the cytokine most commonly and significantly upregulated, irrespective of treatment compound , suggesting that TNF-α is a sensitive and general inflammation marker to evaluate mucosal toxicity induced by different microbicide candidates.The safety profile of a microbicide candidate should be carefully evaluated before moving the candidate into clinical trial. As we mentioned above, the removal of two natural barriers at mucosa will increase the risk to acquire HIV-1 infection, therefore, anti-HIV-1 microbicides should not cause the disruption of mucosa integrity or inflammatory responses. Previous studies demonstrated that the systemic application of SFT was well tolerated without any severe adverse events in Phase Ia clinical trial in vivo, a real-time monitoring will be required to determine its pharmacokinetics. In addition, SFT as an entry inhibitor is likely to exert its synergistic effect with other classes of antiretroviral drugs, such as reverse transcriptase inhibitor and protease inhibitor, and thereby be employed in the development of more potent microbicide candidate.Overall, the present study provides evidences that the SFT gel is a promising vaginal microbicide candidate and could be employed to build up the third barrier at the mucosal site. Though it is speculated that SFT may possess a prolonged half-life


Bacillus subtilis strains by morphological, physiological, and biochemical characterization and 16S rRNA gene analysis. The production patterns of cellulose or hemicellulose-degrading enzymes were investigated during cell culture. All three isolated strains produced CMCase, Avicelase, β-glucosidase, and xylanase enzymes, which suggested synergic cellulolytic systems in Bacillus subtilis. The enzymes showing CMCase, Avicelase, and xylanase activities existed in cell-free culture supernatant, meanwhile β-glucosidase activity was detected in cell debris suggesting that three of the enzymes, including CMCase, Avicelase, and xylanase, were extracellular, and β-glucosidase was cell membrane bound. The three isolates, SL9-9, C5-16, and S52-2, were not the same strains, presenting slight differences in biochemical characteristics, 16S rRNA gene sequences, and cellulolytic enzyme activities.The bioconversion of cellulose and hemicellulose to soluble sugars is important for global stabilization and a sustainable human society. Here, hundreds of cellulolytic bacteria were screened and isolated from soil, compost, and animal waste slurry in Jeju Island, South Korea. Among the isolates, three strains, SL9-9, C5-16, and S52-2, showing higher potential for practical uses were purified on carboxymethyl cellulose (CMC) agar plates and identified as  The bioconversion of cellulose to soluble sugars and glucose is catalyzed by a group of enzymes called cellulases that are produced by microorganisms .These cellulolytic microorganisms play an important role in the biosphere by recycling cellulose, the most abundant and renewable biopolymer on Earth. The demand for microbial cellulases and related enzymes is growing more rapidly than ever before .β-glucosidases (or cellobiases), acting in synergy for the complete hydrolysis of cellulose [Trichoderma viride was first demonstrated by Giligan and Reese [Fungal cellulases are produced in large amounts, which include all the components of a multienzyme system with different specificities and modes of action, that is, endoglucanases (or CMCase), exoglucanases (cellobiohydrolase), and ellulose –6. Synernd Reese .Pseudomonas and Actinomyces, facultative anaerobes such as Bacillus and Cellulomonas, and strict anaerobes such as Clostridium. Most of these bacteria produce mainly endoglucanases [Bacillus species secrete cellulases, including strains of B. cereus [B. subtilis [B. licheniformis [Bacillus sp. KSM-330 [Bacillus [Bacillus endoglucanases (CMCase) that have shown detectable activity on microcrystalline cellulose [Synergic multienzyme systems are also expected in bacterial cellulase complexes. Cellulolytic bacteria include aerobes such as ucanases . A varie. cereus , B. subtsubtilis , B. lichniformis , Bacillu KSM-330 , and alkBacillus . In addiBacillus . Howeverellulose , 15.Bacillus subtilis strains from soil, compost, and animal waste slurry and studied their cellulolytic enzymes. This paper reports the occurrence of these cellulolytic enzymes from Bacillus subtilis strains isolated from different habitats. According to the results, the strains possess microcrystalline cellulose-hydrolytic activity, cell-bound β-glucosidases, and hemicellulases in addition to endoglucanases.We isolated cellulase-producing Bacillus licheniformis KACC10476, B. pumilus KACC10917, and B. subtilis KACC10111, were obtained from Korean Agricultural Culture Collection . Four strains, B. amylolicheniformis KCTC2105, B. licheniformis KCTC3045, B. pumilus KCTC3348, and B. subtilis KCTC3560, were obtained from Korean Collection for Type Cultures . Three bacterial isolates were acquired in this study and deposited in the KACC under registration number KACC91232P for Bacillus subtilis SL9-9, KACC91229P for Bacillus subtilis C5-16, and KACC91233P for Bacillus subtilis S52-2.Three strains, 4)2SO4, 2.5; K2HPO4·3H2O, 0.25; NaCl, 0.1; MgSO4·7H2O, 0.125; FeSO4·7H2O, 0.0025; MnSO4·4H2O, 0.025; agar, 10, and then the plates were incubated at 28°C for 2 days. The incubation temperature focused on mesophiles. The initial medium pH was adjusted to 7.0 if not specified. A few bacterial colonies were harvested and transferred to fresh CMC agar plates containing trypan blue. The plates were incubated at 28°C for 2 days, and the cellulolytic clones were detected by clear halos around the colonies [A total of 176 samples were collected from soil, compost, and animal waste slurry on Jeju Island, South Korea, and were screened for cellulolytic bacteria. The samples were stored at 4°C in the dark until use. After appropriate dilutions with sterile water, 1 mL each of the sample dilutions was spread onto carboxymethyl cellulose (CMC) agar plates that consisted of CMC, 10.0; yeast extract, 1.0; . An API 50 CHB kit  was also used for the identification of Gram-positive bacteria. API strips were inoculated with 24 h-grown cultures and then incubated at 28°C. The results were read according to the manufacturer's instructions and compared with other known ocedures  were useμL of template (50 ng/μL), 5 μL of 10x reaction buffer , 5 μL of deoxynucleoside triphosphates (2.5 mM each), 1 μL of each primer (10 pmol/μL), 0.5 μL of Taq DNA polymerase, and distilled sterile water to make final total volume of 50 μL. The reaction mixture was incubated in a thermocycler  programmed to run 30 cycles repeatedly  and, finally, further incubated at 72°C for 10 min for DNA amplification. The molecular sizes of the resulting PCR products were analyzed on 1.0% agarose gel to confirm 1.5 kb 16S rRNA. This 16S rRNA was purified using a DNA purification kit , and its nucleotide sequences were determined by the dideoxy chain-termination method [Genes of 16S rRNA were sequenced and compared for identification of the bacterial isolates. The bacterial cells grown on CMC agar were harvested and used for chromosomal DNA isolation according to the protocols . The chrn method  using a Starter cultures were prepared by transferring cells with an inoculation loop from the CMC agar plates to 100 mL of CMC liquid medium, the initial pH of which was adjusted to 7.0 if not specified, in 500 mL Erlenmeyer flasks. Two days after shaking incubation at 28°C, aliquots of 2 mL starter cultures were seeded into 200 mL of CMC liquid medium in 500 mL flasks. The flasks were further incubated on a shaker at 150 rpm for 7 days at 28°C. Cell growth was monitored by measuring optical density at 600 nm. Culture samples were taken every 24 h during incubation, and their cell-free supernatants (CFSs) were obtained by centrifugation  and analyzed for cellulolytic activities. Meanwhile, the precipitated cells were suspended, washed in 5 mL of 0.05 M phosphate buffer (pH 6.5), and disrupted by sonication . The resulting supernatant was removed after centrifuging  the sonicated cell suspension at 4°C. The remaining cell debris (CD) was resuspended in 1 mL of 0.05 M phosphate buffer (pH 6.5) and assayed for cellulolytic enzyme activities.μmol of reducing sugars as glucose equivalents min−1.CMCase activity was measured by incubating 0.2 mL of enzyme solution with 0.5 mL of 1% (w/v) carboxymethyl cellulose, prepared in 0.1 M sodium acetate buffer (pH 5.0), and 0.3 mL of 0.1 M sodium acetate buffer (pH 5.0) for 10 min at 50°C. The reducing sugars liberated were estimated by the 3,5-dinitrosalicylic acid (DNS) method . The enzμmol of reducing sugars as glucose equivalents min−1. Avicelase activity was measured by incubating 0.5 mL of enzyme solution with 1 g of Avicel, a microcrystalline cellulose, as substrate and 1.5 mL of 0.1 M sodium acetate buffer (pH 5.0) for 1 h at 50°C. After incubation, the reaction mixture was centrifuged at 10,000 ×g for 5 min, and then 1 mL of the supernatant was taken to determine reducing sugars by the DNS method. One unit of Avicelase activity was defined as the amount of enzyme that released 1 μmol of reducing sugars as glucose equivalents min−1.Filter paper-hydrolytic (FPase) activity was measured by a procedure  in whichβ-glucosidase (or cellobiase) activity was measured by using ρ-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate. The hydrolysis of pNPG releases ρ-nitrophenol, a pigmented substance that can be measured spectrophotometrically at 400 nm. The reaction mixture, containing 0.5 mL of 1 mg/mL pNPG, 0.5 mL of 0.05 M sodium acetate buffer (pH 5.0), and 0.5 mL of enzyme solution, was incubated at 50°C for 1 h. The enzyme reaction was stopped by adding 2 mL of 1 M Na2CO3, and the absorbance was measured at 400 nm. One unit of β-glucosidase activity was defined as the amount of enzyme that released 1 μmol of para-nitrophenol min−1.μmol of reducing sugars as xylose equivalents min−1.Xylanase activity was measured by using Beechwood xylan as a substrate . The reaBacillus species obtained from KACC and KCTC. Finally, three clones showing relatively higher cellulolytic activity and broader pH optimum were selected . The isolates C5-16 (accession no. HQ236380) and S52-2 (accession no. HQ236381) showed the highest identity at 99% with Bacillus subtilis strain CM19 (accession no. EU660332.1) and at 100% with Bacillus subtilis isolate C9-1 (accession no. EU257446.1), respectively. Based on their morphological, physiological, and genetic data, the three bacterial isolates were designated as Bacillus subtilis SL9-9, C5-16, and S52-2, respectively.These three bacterial isolates were finally identified by 16S rRNA gene sequence analysis. Their sequences were entered into the nucleotide-nucleotide BLAST (NCBI) system, and percentage identities were established. The highest identity for the isolate SL9-9 (accession no. HQ236379) was 99% with the β-glucosidase, FPase, and xylanase production after cultivation in 200 mL of CMC liquid medium. Bacillus subtilis KACC10111, which showed higher CMCase activity than the other 6 Bacillus species obtained from KACC and KCTC  were observed and slightly increased as cultivation continued like the Avicelase activity profiles (data not shown). It is highly possible that both the Avicelase and CMCase  activity resulted from the same enzyme protein. Our results show some contrast to a previous report [β-1,4-glucanase produced by B. subtilis DLG was not able to significantly degrade crystalline cellulosic substrates. Fukumori et al. [β-1,4-glucanases from alkalophilic B. subtilis strains 1139 and N-4 were capable of hydrolysing CMC, but could not degrade Avicel significantly. Hamamoto et al. [2-terminus and COOH-terminus of the endoglucanase in Clostridium cellulovorans is essential for the hydrolysis of crystalline cellulose. These phenomena suggest that crystalline cellulose-hydrolyzing activity does not depend on the same catalytic site of endo-β-1,4-glucanase.On a whole, SL9-9 presented higher activity than the other isolates from the beginning of cultivation. In the cell debris fraction, there was no definite Avicelase activity . The pros report  in whichi et al.  also repβ-glucosidase (or cellobiase) activity profiles obtained during shaking incubation for 7 days with 10 g/L of carboxymethylcellulose as a carbon source. In contrast to CMCase and Avicelase, no β-glucosidase activity was observed in the cell-free supernatant. However, all the strains showed considerable β-glucosidase activity in the cell debris fraction. Maximum activities  were detected early after cultivation for 24 h. The enzyme activities subsequently decreased after reaching maximum values, and then second rises and falls were observed. A reason for the rise and fall in β-glucosidase activity might be the negative regulation of β-glucosidase gene expression by glucose level (catabolite repression) in the cells. β-glucosidase activities are inferred to be related to membrane-associated enzymes. So far, β-glucosidase has scarcely been reported in Bacillus strains, although its production by other bacteria like Clostridium thermocellum [Alcaligenes faecalis [β-glucosidase in actinomycete Microbispora bispora was cell membrane bound. Pajni et al. [Bacillus species isolated from soil produced xylanase, and 82.4% of them also produced β-glucosidase. On the other hand, Dhillon et al. [B. licheniformis could grow in minimal media containing cellobiose, but failed to show the presence of cellobiase in either the cellular fraction or culture supernatant. It was hypothesized that the utilization of cellobiose even in the absence of cellobiase involved the enzyme cellobiose phosphorylase [mocellum  and Alcafaecalis  has beenfaecalis  reportedi et al.  reportedBacillus strains SL9-9 and S52-2 showed considerable xylanase activity in the cell-free culture supernatant, and their activities reached maximum values  after shaking culture for 96 h with 10 g/L of carboxymethylcellulose as a carbon source, as shown in Bacillus strains [Bacillus species were also xylanase positive, and units of xylanase activity were found to be much higher as compared to corresponding CMCase activity units. Xylans, with a linear backbone of β-1,4-linked xylose residues, form the major group of hemicelluloses. Endoxylanases hydrolyze xylan to xylooligosaccharides and xylose residues, while β-xylosidases catalyze the release of xylosyl residues by the terminal attack of xylooligosaccharides. It is highly possible that the xylanase activities of our Bacillus subtilis strains came from the combined actions of independent endoxylanase and β-xylosidase enzymes.The  strains . Pajni e strains  reportedBacillus subtilis in this study. The isolates were not the same strains, showing slight differences in biochemical characteristics, 16S rRNA gene sequences, and production patterns of cellulases and xylanases. They had microcrystalline cellulose-hydrolytic activity in addition to β-glucosidase, hemicellulase, and endoglucanase activities. These strains are presently being employed in agriculture as a fertilizer supplement. They especially were quite effective as ingredients of an organic seedbed.Three cellulolytic bacterial strains, SL9-9, C5-16, and S52-2, were isolated and identified as


Bifurcaria bifurcata, and the evaluation of the antioxidant and antimicrobial activity of bacteria extracts. The identification of epiphytic bacteria was determined by 16S rRNA gene sequencing. Bacteria extracts were obtained with methanol and dichloromethane (1:1) extraction. The antioxidant activity of extracts was performed by quantification of total phenolic content (TPC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Antimicrobial activities were evaluated against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella enteritidis, Staphylococcus aureus, Saccharomyces cerevisiae and Candida albicans. A total of 39 Bifurcaria bifurcata-associated bacteria were isolated and 33 were identified as Vibrio sp. (48.72%), Alteromonas sp. (12.82%), Shewanella sp. (12.26%), Serratia sp. (2.56%), Citricoccus sp. (2.56%), Cellulophaga sp. (2.56%), Ruegeria sp. (2.56%) and Staphylococcus sp. (2.56%). Six (15.38%) of the 39 bacteria Bifurcaria bifurcata-associated bacteria presented less than a 90% Basic Local Alignment Search Tool (BLAST) match, and some of those could be new. The highest antioxidant activity and antimicrobial activity (against B. subtilis) was exhibited by strain 16 (Shewanella sp.). Several strains also presented high antimicrobial activity against S. aureus, mainly belonging to Alteromonas sp. and Vibrio sp. There were no positive results against fungi and Gram-negative bacteria. Bifurcaria bifurcata epiphytic bacteria were revealed to be excellent sources of natural antioxidant and antimicrobial compounds.Surface-associated marine bacteria are an interesting source of new secondary metabolites. The aim of this study was the isolation and identification of epiphytic bacteria from the marine brown alga,  The marine environment is extremely complex and contains a huge diversity of life forms, which is a huge source of biological and chemical diversity ,2,3. TheAmong marine organisms, macro-algae can host varieties of heterotrophic bacteria, and many of these bacteria play an important role in maintaining the health of the host organism by producing unique bioactive secondary metabolites. With the increasing need for novel drug discovery, an understanding of the marine epiphyte-host associations outlined above is likely to deliver a rich source of preexisting and of new high value-added biomolecules with the potential for providing sustainable economic and human benefits ,9,16,17.Bifurcaria bifurcata is a perennial brown alga, found abundantly along the North East Atlantic shores, from Ireland as the northern limit, to south of the Western Sahara as the southern limit [Bifurcaria bifurcata extracts [Bifurcaria bifurcata and the evaluation of the antioxidant and antimicrobial activities of extracts obtained from the isolated strains.rn limit . Previouextracts ,20. Howe6 bacterial cells per milliliter [Bifurcaria bifurcata surface were isolated, and the strains were subjected to molecular identification using 16S rRNA gene sequencing.The oceans are massively complex and consist of diverse assemblages of life forms. The water column of the oceans contains approximately 10lliliter . In thisVibrio sp. was the most common bacteria, representing 48.72% of all the B. bifurcata isolated bacteria. Bacteria belonging to the Vibrionaceae family are widespread in the marine environment. This family is particularly abundant on the surface of marine macroorganisms, where they form commensal, symbiotic or pathogenic associations [Alteromonas sp. and Shewanella sp. were the following more representative genus, with 12.82% and 12.26% of occurrence, respectively. Both strains are frequent in the marine environment and have already been found to be associated with marine invertebrates, namely sponges and algae [Cellulophaga sp., Serratia sp., Ruegeria sp., Staphylococcus sp. and Citricoccus sp. were represented by one strain each (2.56%). However, these bacteria are found to be frequently associated with marine invertebrates and have been the focus of study by other authors [As shown in ciations . Alteromnd algae ,24. Cell authors ,26,27,28Marine bacteria develop unique metabolic and physiological capabilities, which enable them to survive in extreme habitats and to produce compounds that might not be produced by their terrestrial counterparts ,6.Bifurcaria bifurcata, and screened for their antioxidant and antimicrobial capacities. Since there is not a specific method to evaluate the antioxidant activity of a compound/extract, due to different antioxidant mechanisms, the antioxidant activity was evaluated through three methods, namely, oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity and total phenolic content (TPC).In this work, marine bacteria were isolated from the brown alga, Shewanella genus, showed the highest phenolic content (53.854 ± 3.02 GAE/g extract) followed by Bacteria 8 (14.222 ± 4.25 GAE/g extract) and 36 (10.376 ± 1.58 GAE/g extract). There are not many works quantifying the total phenolic content of marine bacteria, especially epiphytic algae. However, a total phenolic content of 0.22 mg GAE/g of extract was found on the marine Actinobacteria, which was significantly lower when compared with the results obtained by the B. bifurcata-associated bacteria study [The antioxidant results are stated in ia study . Moreoveia study .Shewanella sp.) and Bacteria 39 (Vibrio sp.), which showed an IC50 of 20.21 (14.41–28.34) µg/mL and 23.6 (19.5–28.7) µg/mL, respectively. The Bacteria 8, 11, 26 and 36 also denoted quite high antioxidant activity on the reduction of DPPH with an IC50 of 164 (109–247) µg/mL, 183.8 (118.5–285.2) µg/mL, 521 (366.9–739.7) µg/mL and 587.7 (442–781.4) µg/mL, respectively. These results are extremely interesting, especially when compared with the synthetic antioxidant, BHT (butylated hydroxytoluene), which presented an IC50 of 40.55 (27.39–60.05) µg/mL, since Bacteria 16 and 39 presented more potency in scavenging the DPPH radical. Kalirajan and co-workers [50 of 857.49 µg/mL, which is 40 times less potent than that obtained for Bacteria 16 and 39 in our work.Regarding the DPPH radical scavenging activity, the highest potency was produced by Bacteria 16 . Once more, Bacteria 16 showed the highest antioxidant activity with an ORAC value of 3603.659 ± 53.38 µmol of TE/g extract, followed by Bacteria 8, 31, 23 and 26 with 912.621 ± 23.79 µmol of TE/g extract, 692.260 ± 13.61 µmol of TE/g extract, 561.990 ± 12.69 µmol of TE/g extract and 520.917 ± 14.13 µmol of TE/g extract, respectively. The lack of information about the production of antioxidant compounds produced by marine epiphytic bacteria, namely evaluated by the ORAC method, does not allow us to make a direct comparison with other works; however, Wang and co-workers [Recently, the ORAC assay has received much attention as a new orescein . Accordiorescein , the ORA-workers  evaluateB. bifurcata epiphytic bacteria presented in this work can be explained by the environmental conditions where these bacteria exist. They live in close association with soft-bodied marine organisms, which lack obvious structural defense mechanisms, and, thus, rely on chemical defense by the production of bioactive secondary metabolites to survive in their extreme habitat [The high antioxidant activity of  habitat . The pro habitat .Shewanella (Bacteria 16), produces long-chain polyunsaturated fatty acids, and recent studies associated these compounds with an efficient antioxidant system mechanism, which operates as a primary protective “breakwater” for all marine microorganisms possessing them [Shewanella sp.).It is well known that the genus, ing them . This caBesides the production of antioxidant compounds, marine bacteria also have the ability to produce antimicrobial compounds that play an important role in the protection against other pathogenic and fouling microorganisms .B. bifurcata epiphytic bacteria was also evaluated against several microorganisms, including human pathogens, namely Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Saccharomyces cerevisiae, Salmonella enteritidis and Bacillus subtilis. Extracts were tested against all the mentioned microorganisms at 1 mg/mL; the ones that presented a capacity to inhibit the microorganism’s growth in more than 50% (IC50 < 1 mg/mL) were also evaluated through dose-response analysis , and the IC50 was determined. The B. bifurcata-associated bacteria extracts did not show antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Saccharomyces cerevisiae and Salmonella enteritidis (data not shown). However, several of the B. bifurcata-associated bacteria extracts induced high antibacterial activity against Bacillus subtilis and Staphylococcus aureus (50 ranged from 2.29 µg/mL (1.79–2.94) to 621.4 µg/mL (508.2–759.9) exhibited by Bacteria 16 (Shewanella sp.) and 13 (Vibrio sp.), respectively, against B. subtilis, and 50.85 µg/mL (40.72–63.50) to 722.9 µg/mL (541.11–965.9) exhibited by Bacteria 34 (Alteromonas sp.) and 4 (Cellulophaga sp.), respectively, against S. aureus. Actually, the potency of some bacteria extracts against B. subtilis growth, such as Bacteria 16 (Shewanella sp.), 25 (Shewanella sp.), 17 (Shewanella sp.), 34 (Alteromonas sp.) and 24, was even higher than that of the commercial antibiotic, chloramphenicol. Moreover, the B. bifurcata-associated bacteria extracts from the Bacteria 13B (Serratia sp.), 39 (Vibrio sp.), 50 (Vibrio sp.) and 38 had a chloramphenicol similar potency.In this work, the antimicrobial activity of compounds produced by s aureus . The obtAlteromonas genus, showed the highest antimicrobial activity against Staphylococcus aureus with an IC50 of 50.85 and 43.48 µg/mL, respectively. In line with our results, Barja and co-workers [Alteromonas sp. that exhibited a broad inhibition, including against Staphylococcus aureus. Furthermore, Shiozawa and co-workers isolated a new antibiotic also produced by Alteromonas sp. with high antibacterial activity [Alteromonas sp. isolated from a marine invertebrate also exhibited high antimicrobial activity, namely against fish pathogenic bacteria [The extracts from Strains 34 and 6, which belong to the -workers  isolatedactivity . In anotbacteria .Bacillus are current in the marine environment. For instance, Bacillus licheniformis (strain EI-34-6) and Bacillus subtilis (strain II-111-5) were isolated from the marine alga, Palmaria palmate [Bacillus strains are direct competitors of other species of marine epiphytic bacteria, like Shewanella sp., Alteromonas sp., Serratia sp. and Vibrio sp., which can explain the high antimicrobial activity of these bacteria’s extracts against Bacillus subtilis. In line with these findings, it was not surprising that more than 50% of the total bacteria isolated from the B. bifurcata macro-alga presented antibacterial activity against B. subtilis.It is widely accepted that marine organisms produce compounds to survive and adapt to the unfavorable conditions where in which they live, and the production of antimicrobial compounds by marine bacteria seems to be a powerful weapon in space competition, namely for surfaces colonization. Other strains of  palmate . TherefoAccording to the literature, the production of antimicrobial compounds by marine organisms against Gram-positive bacteria seems to be more frequent . The genShewanella sp.), which revealed huge antioxidant and antimicrobial activities.In this study, some bacterial extracts revealed positive bioactivity results, and given that these are multi-component extracts, there is a potential for discovering multiple active compounds in a single fraction, making these organisms a valuable source of novel substances for future marine drug discovery. The very high antioxidant and antimicrobial activities observed could be associated with low-abundant substances, indicating a high potency for these compounds, especially for Bacteria 16 , which were incubated at 20 °C. Colonies were selected at random and by systematic sampling, with distinctive colony types being selected along with a random sample from each plate. For purifying bacteria, we performed the streak technique at least 5 times.Marine alga ca. 1500 bp in length were amplified using the bacterial universal primers, F27 (5′-AGAGTTTGATCMTGGCTCAG-3′) and R1492 (5′-TACGGYTACCTTGTTACGACTT-3′) [® Flexi DNA Polymerase Buffer , 3 µL of MgCl2, 25 mM, 2.5 µL dNTPs, 2 mM, 1.5 µL of each primer, 10 µM, 1.25 of dimethyl sulfoxide (DMSO) 100%, 0.5 µL GoTaq® Flexi DNA Polymerase 5U/µL and 1 µL of DNA template. Thermal cycling started with an initial denaturation step of 94 °C for 3 min, 35 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 72 min and a final extension step of 72 °C for 10 min. All PCR amplifications were carried out in a Gradient Thermal cycler . All amplicons were checked under UV light after electrophoresis in 1.5% agarose gels stained with RedSafe™ . PCR products with the right size (ca. 1500 bp) were cleaned with DNA Clean & Concentrator™-5  columns and subjected to sequencing with the Sanger method in a BigDye Terminator 3  using the forward and reverse primer. Nearly complete 16S rRNA gene sequences were obtained by sequencing with the forward and reverse primers. All sequences were automatically trimmed using the Phred quality score method [DNA was extracted from previously stored bacterial pellets with the GeneJET™ Genomic DNA Purification Kit , according to the manufacturer’s instructions. Genus-level identification of isolates was determined by 16S rRNA gene sequencing. 16S rRNA gene fragments of e method . Their c4, 0.1 g; dissolved in 1 L seawater; pH 7.2–7.6) and incubated with aeration for three days at 25 °C. Bacteria cells were centrifuged and the pellet recovered and lyophilized. Lyophilized cells were then extracted with methanol and dichloromethane (1:1) at constant stirring for 12 h. The solvents were evaporated in a rotary evaporator at 40 °C, and the extracts were solubilized in dimethyl sulfoxide (DMSO) and stored at −20 °C until further use.Isolated bacteria were cultured in 500 mL of Marine Broth  and used to calculate the phenolic content using gallic acid as the standard.The TPC of bacteria extracts was determined using the Folin-Ciocalteu method adapted to the microscale . Briefly® Instruments). The radical solution was freshly prepared each day. The synthetic antioxidant, BHT, was used as the standard. The ability to scavenge the DPPH radical was calculated using the follow equation:control is the absorbance of the control (198 µL of DPPH solution plus 2 µL DMSO), Asample is the absorbance of the test sample (198 µL of DPPH solution plus 2 µL sample) and Asample blank is the absorbance of the sample in methanol (198 µL of MeOH plus 2 µL of sample).The DPPH free radical scavenging method was performed according to Brand-Williams (1995)  adapted The oxygen radical absorbance capacity assay (ORAC) method was performed as described by Dávalos and co-workers  as folloversus time) were first normalized to the curve of the blank corresponding to the same assay by multiplying original data by the factor fluorescencet=0blank, /fluorescencet=0sample, . From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated as:f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at time i. The net AUC corresponding to a sample was calculated by subtracting the AUC corresponding to the blank. Regression equations between net AUC and antioxidant concentration were calculated for all the samples. ORAC values were expressed as micromoles of Trolox equivalent/gram of extract by using the standard curve calculated for each assay.Antioxidant curves (fluorescence Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633) and Salmonella enteritidis (ATCC 13076) cultured in Luria broth (LB); Staphylococcus aureus (ATCC 25923) cultured in trypticase soy yeast extract medium (TSYE); and Saccharomyces cerevisiae (ATCC 9763) and Candida albicans (ATCC 10231) cultured in yeast extract peptone dextrose (YPD) medium. All mediums were obtained from Merck .The antimicrobial activity of bacteria extracts was evaluated against seven microorganisms: 600)) during 48 h (data not shown) to determine the beginning of the exponential phase, in which the effect of bacterial extracts compared with the vehicle (microorganisms growth in the presence of 1% DMSO) will be assessed.Microorganisms growth was monitored spectrophotometrically . Results were expressed in the percentage of control.The antimicrobial assays were performed in 96-well plates, where 193 µL of medium, 5 µL of microorganism inoculum and 2 µL of extracts samples were added per well. Bacteria were incubated at 37 °C and fungi at 30 °C. As positive controls, ampicillin (0.01–1 µg/mL), amphotericin b (1–100 µg/mL), bacitracin (0.1–10 µg/mL), chloramphenicol (1–100 µg/mL) and oxytetracycline (0.01–1 µg/mL), purchased from Sigma Aldrich , were used, previously prepared in sterile-filtered DMSO and stored at −20 °C. The ability of bacteria extracts to inhibit the microorganisms growth was accompanied by optical density at 600 nm (X − LogIC).Results are presented as the mean ± standard error of mean (SEM). The ICBifurcaria bifurcata-associated bacteria were isolated and identified. This study reveals that B. bifurcata epiphytic bacteria are an excellent source of new natural antioxidant compounds, as well as antimicrobial compounds against Gram-positive bacteria. Moreover, all bacteria presented in this work are cultivable in a flask scale, which opens an opportunity for the production of the bioactive compounds in an industrial scale. As the production of secondary metabolites is related to environmental factors, such as UV radiation, pH, temperature, nutrient availability and time of fermentation, these can be manipulated in order to expose the bacteria to stress situations eliciting the production of the target bioactive compound. From a biotechnological perspective, the production of secondary metabolites, as effective competition and defense strategies by surface-associated microorganisms, constitutes an unparalleled reservoir for the discovery of new drugs, therapeutic agents and bioactive molecules, with applications across medical, industrial and environmental settings.To the best of our knowledge, for the first time,


Alphaproteobacteria, Gammaproteobacteria, Bacteroidetes and Actinobacteria. Only seven isolates were psychrophilic, 15 isolates were moderately psychrophilic, and 11 isolates were psychrotolerant. More than 72% of the isolates required sodium chloride to grow. Esterase, β-glucosidase and proteases activities at 4 °C were detected in more than 45% of the strains while approximately 21%, 15% and 12% of the strains possessed lipase, amylase and chitinase, respectively. These results indicate that a relatively high culturable bacterial diversity is present within marine sediment of Nella Fjord and it could serve as an ideal candidate region for bioprospecting. The diversity and cold-active hydrolytic enzymes of culturable bacteria associated with sandy sediment from Nella Fjord, Eastern Antarctica  was investigated. A total of 33 aerobic heterotrophic bacterial strains were isolated at 4 °C. These bacterial isolates could be sorted into 18 phylotypes based on the 16S rRNA gene sequence belonging to four phyla, namely  Nella Fjord, located off the west coast of Bruknes Peninsula, Eastern Antarctica, is a U-shaped inlet whose narrow neck is nearly totally blocked with smaller icebergs. There, the ice cover is present for no less than ten months a year. Nella Fjord has attracted the attention of scientists as a long-term ecological monitoring site for evaluating polar ecosystems during climatic change . EarlierIn this study, we present the phylogenetic analysis of 33 bacterial isolates in order to obtain a preliminary understanding of the bacterial community composition in the sediment of Nella Fjord. In addition, these microorganisms were also used for bioprospecting for cold-active hydrolytic enzymes, which have numerous potential applications in biotechnology processes .4 cfu/g sediment. The growth temperature ranges of all strains were tested on marine 2216 agar plates. After four weeks of incubation, growth of all strains occurred at 0 °C and no growth at 42 °C. Only seven strains were psychrophilic showing no growth above 20 °C [bona fide marine organisms and are well adapted to the low temperature and relatively constant salinities associated with the marine sediment from Nella Fjord. A total of 33 bacterial strains were isolated from sandy marine sediment sampled from Nella Fjord using aerobic heterotrophic conditions at 4 °C. The bacterial number in the sample was 1.3 ± 0.5 × 10ve 20 °C , and 15 ve 20 °C   [Sphingopyxis sp. KBO-5 from Kongsfjorden marine sediment [Out of the 17 Gram-negative phylotypes, 10 belonged to the phylum tulatus) . Furthersediment , suggestsediment . Additiosediment . Colwellia, Marinomonas, Moritella, Photobacterium, Bizionia and Salinibacterium isolates. The ability to degrade esculin was almost equally distributed among the isolates of Gammaproteobacteria and Bacteroidetes, but not Alphaproteobacteria and Actinobacteria. Skim milk was also hydrolyzed by most of our strains at 4 °C.However, Sphingopyxis, Glaciecola, Marinomonas and Marinobacter isolates did not produce proteases. Lipase activity was detected in members of Sulfitobacter, Colwellia, Glaciecola, Photobacterium and Pseudomonas. Chitin was exclusively hydrolyzed by the members of Shewanella. Amylase positive isolates were members of Colwellia, Photobacterium, Pseudomonas and Bizionia. The three β-galactosidases producers were distributed among the members of Marinomonas, Shewanella and Flavobacterium.The production of hydrolytic enzymes at 4 °C by the 33 isolates is summarized in The ability of the newly isolated psychrophilic or psychrotolerant strains to produce a broad spectrum of cold-active enzymes is of great interest for both fundamental research and industrial applications. The high number of esterase-producing, β-glucosidase-producing and/or protease-producing bacteria among the isolated strains  could beetc.) [Diverse cold-active enzymes detected in this study may find applications in various industries  . Cold-acin situ temperature of the sample was −1.5 °C, and the in situ salinity was 35‰. This sediment sample was stored in sterilized plastic bags (250 mL) and transported to the laboratory at temperatures between 0 and 4 °C. The sandy marine sediment sample was collected from the Nella Fjord, Prydz Bay, Eastern Antarctica  at a water depth of 20 m by Boris I. Sirenko from the Zoological Institute of Russian Academy of Sciences when he dived for investigation of the benthic fauna on 12 January 2007. The In the laboratory, one gram of wet sediment sample was mixed with 99 mL of sterilized seawater supplemented with 10 glass beads (Diameter 2-3 mm) shaken at 4 °C for 1 h at 300 repetitions/min. The suspension was further diluted (1:10) in the sterilized seawater and spread onto three different marine agars. These included 1/10-strength marine R2A agar , 1/10-st3.3. 16S rDNA Amplification, Sequencing Escherichia coli DH5α competent cells. Recombinants were selected using Luria-Bertani (LB) indicator plates containing 100 µg of ampicillin per mL, 80 µg of X-Gal  per mL, 0.5 mM IPTG . White clones were sequenced by an ABI PRISM 3730 sequencer at Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Total genomic DNA for 16S rDNA amplification was isolated from 1 mL of bacteria grown to late log phase in marine 2216 broth (Difco) and purified by kit according to the manufacturer’s instruction . Nearly full-length 16S rRNA gene was amplified by PCR using universal primers 8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492r (5′-GGT TAC CTT GTT ACG ACT T-3′) . The PCRThe sequences, 1427 nucleotides (nt) to 1515 nt, depending on the isolate, were compared with the data available in the RDPII  using the sequence match tool, to determine the relative phylogenetic positions. The identification of phylogenetic neighbors and the calculation of pairwise 16S rDNA sequence similarities were achieved using the EzTaxon server . In addi2, 2 g MgSO4, 0.5 g CaCl2, 1 g KCl, 5 g peptone and every concentrations of NaCl at pH 7.5, adjusted with KOH [Growth of isolates in a temperature range of 0-42 °C was studied on marine 2216 agar. The strains were cultivated at 0, 4, 10, 15, 20, 25, 30, 37 and 42 °C for 2 to 4 weeks. Growth was monitored at up to 64× magnification by using a Leica stereoscope . The tolerance for NaCl  was tested in medium containing (1 000 mL deionized water) 5 g MgClProtease, esterase, lipase, chitinase, amylase, cellulase, β-glucosidase and β-galactosidase were detected on diagnostic plates after incubation at 4 °C for 2 to 4 weeks. Protease activity was tested on skim milk plates containing (per liter NSW) 50 g of skim milk, 5 g of peptone and 1 g of yeast extract. Clearing zones around the colonies were used as an indication of protease activity. Esterase activity was detected on tributyrin plates containing (per liter NSW) 5 mL of tributyrin, 5 g of peptone and 1 g of yeast extract. Formation of clearing zones around colonies was used to indicate esterase activity. Lipase activity was detected by using Rhodamine B plus olive oil agar plates, as described previously . Chitin The 33 bacterial strains categorized into 18 phylotypes indicated that a relatively high diversity was present within the bacterial community associated with sandy marine sediment from Nella Fjord, Eastern Antarctica. A vast variety of cold-active biopolymer-degrading enzymes detected in this study indicated that isolated bacteria could contribute significantly to the hydrolysis of the major organic constituents  and are therefore involved in carbon cycling and nitrogen cycling in the sediment of Nella Fjord. Furthermore, the data obtained in this study also confirmed that Nella Fjord could serve as an ideal candidate region for bioprospecting.