Effects of Swimming Pool Conditions and Floor Types on White Roman Geese's Physical Condition Scores and Behaviors in an Indoor Rearing System.

Biosecurity problems, including the continual risk of avian influenza spread by wild birds, have severely affected traditional free-range waterfowl production systems. Regulations and techniques for indoor goose production require more considerations for animal welfare. This study investigated the effects of swimming pool conditions and different floor types on the physical condition scores and behaviors of indoor-reared White Roman geese. A total of 48 male and 48 female White Roman geese reared from the age of 15 to 84 days were randomly allocated to pens with or without a swimming pool and with either mud or perforated plastic floors. Providing a swimming pool improved geese's eye and feather cleanliness and breast blister scores at the age of 84 days. Compared with geese reared on a mud floor, those reared on a perforated plastic floor had better feather cleanliness and higher breast blister scores at the age of 56 and 84 days. Providing a swimming pool to indoor-reared geese may reduce the proportion of abnormal behaviors, such as injurious feather pecking, by increasing water-related behaviors. This study suggests a more appropriate environment design for better balancing commercial goose production with animal welfare in an indoor rearing system.

Novel potent anti-STEAP1 bispecific antibody to redirect T cells for cancer immunotherapy.

BACKGROUND: The prognosis for metastatic Ewing sarcoma family of tumors (EFT) is still poor despite high-dose chemotherapy and radiation treatment. Immunotherapies hold promise, but cancer antigen-targeting immunotherapies have largely failed to induce effective T cell receptor-mediated antitumor response. However, T cell-engaging bispecific antibodies (T-BsAbs) have yet to be adequately explored. METHODS: Rehumanized STEAP1-IgG was used to build T-BsAb (named BC261) using the 2+2 IgG-[L]-scFv platform carrying the anti-CD3 huOKT3 scFv as the second specificity. Its binding epitope mapping, species cross-reactivity, tumor cell line staining, and in vitro cytotoxicity were investigated thoroughly. Its potency in driving tumor-infiltrating lymphocytes (TILs) was quantified using bioluminescence, correlated with in vivo antitumor response against cell line-derived or patient-derived xenografts (CDXs or PDXs) and compared with anti-STEAP1 T-BsAbs built on representative antibody platforms. RESULTS: BC261 binding epitope was mapped to its second extracellular domain of STEAP1 shared among canine and primate orthologs. BC261 induced potent cytotoxicity against panels of EFT, prostate cancer, and canine osteosarcoma cell lines despite their low antigen density. BC261 drove significantly more TILs into tumors (30-fold) and exerted superior antitumor effects compared with the other standard BsAb platforms. The antitumor efficacy of BC261 was consistent against EFT and prostate cancer CDXs and PDXs. CONCLUSIONS: BC261 was highly efficient in driving T cell infiltration and tumor ablation. Either as stand-alone therapeutics or for ex vivo armed T cells, this novel anti-STEAP1 T-BsAb BC261 has therapeutic potential.

Effects of Different Swimming Pool Conditions and Floor Types on Growth Performance and Footpad Dermatitis in Indoor-Reared White Roman Geese.

Footpad dermatitis (FPD) is a major foot disease in modern poultry production, and it affects both poultry health and animal welfare. It refers to inflammation and necrotizing lesions on the plantar surface of the footpads and toes. We investigated the effects of providing a swimming pool and different floor types on growth performance and FPD score in indoor-reared White Roman geese. Forty-eight male and 48 female White Roman geese were randomly allocated to pens with or without a swimming pool and with either mud or perforated plastic floor and reared from 15 to 84 days of age. Growth performance measurements included feed intake (FI), weight gain (WG), and feed conversion ratio (FCR). FI, WG, and FCR were significantly decreased at various growth periods in geese provided with a pool. Lower WG and bodyweight for the perforated plastic floor group were found at 15-28 and 28 days of age, respectively. The geese reared on the perforated plastic floors without a pool had higher FPD scores at 70 and 84 days of age than those with other rearing conditions. A higher incidence of FPD score 1 was observed in geese raised without a pool. In conclusion, providing a pool can improve footpad health in indoor-reared White Roman geese but may not benefit growth performance.

Effects of user experiences on continuance intention of using immersive three-dimensional virtual reality among institutionalized older adults.

AIM: To evaluate the acceptance of three-dimensional virtual reality programs and to explore the factors influencing the acceptance of the programs among the institutionalized older adults. DESIGN: A cross-sectional explanatory study. METHODS: A total of 71 residents completed the program successfully. They were invited to join a 9-week program included eight activities related to horticultural therapy in a virtual environment. Data were collected by structured questionnaires from August 2018 to February 2019. Ten association hypotheses were derived from the literature review. Partial least squares structural equation modelling was used to examine the proposed hypotheses. RESULTS: Program acceptance was defined as virtual reality practices and continuous usage intention. Frequency of practice was about 12 times during 9-week free-trial period, and the score of continuance usage intention was 13.06 (maximum value of 15). The findings indicated that virtual reality practices were significantly affected by presence and the presence was significantly affected by involvement and interactivity. Continuance usage intention was significantly affected by involvement; and involvement was significantly affected by interactivity. However, continuance usage intention was not significantly influenced by virtual reality practices. CONCLUSIONS: The findings supported that immersive three-dimensional virtual reality program was accepted by the institutionalized older adults. The acceptance, in terms of virtual reality practices and continuous usage intention, was influenced by different paths. The findings have a potential impact on the design of virtual reality technology for the care of institutionalized older adults. IMPACT: What problem did the study address? There were controversial findings about the acceptance of immersive 3D virtual reality program among older adults. What were the main findings? Interactivity advanced the sense of presence and, thus, resulted in a higher frequency of virtual reality practices. Interactivity enhanced perception of involvement, thus, contributing to an increased intention of continuous usage Where and on whom will the research have an impact? The immersive 3D virtual reality program was appropriate for older adults, even for first time users or individuals aged over 80 years. The findings revealed new insights for developing immersive 3D virtual reality programs for the older adults.

Effects of a Combination of Three-Dimensional Virtual Reality and Hands-on Horticultural Therapy on Institutionalized Older Adults' Physical and Mental Health: Quasi-Experimental Design.

BACKGROUND: Institutionalized older adults have limited ability to engage in horticultural activities that can improve their physical and mental health. OBJECTIVE: This study explored the effects of a combination of 3D virtual reality and horticultural therapy on institutionalized older adults' physical and mental health. METHODS: The study used a quasi-experimental design. A total of 106 older adults from 2 long-term care facilities were recruited and assigned to the experimental (n=59) or control (n=47) group. The experimental participants received a 9-week intervention. Both groups completed 3 assessments: at baseline, after the intervention, and 2 months later. The outcome variables included health status, meaning in life, perceived mattering, loneliness, and depression. RESULTS: The experimental group demonstrated significantly improved health status (P<.001), meaning in life (P<.001), and perceived mattering (P<.001) as well as significantly reduced depression (P<.001) and loneliness (P<.001) compared to the control group immediately after the intervention; these effects persisted for up to 2 months. CONCLUSIONS: This study verified the beneficial effects of a combination of 3D virtual reality and hands-on horticultural therapy on older adults' health. These results could support the future successful implementation of similar programs for institutionalized older adults on a larger scale.

Collocation of avian and mammal antibodies to develop a rapid and sensitive diagnostic tool for Russell's Vipers Snakebite.

Russell's vipers (RVs) envenoming is an important public health issue in South-East Asia. Disseminated intravascular coagulopathy, systemic bleeding, hemolysis, and acute renal injury are obvious problems that develop in most cases, and neuromuscular junction blocks are an additional problem caused by western RV snakebite. The complex presentations usually are an obstacle to early diagnosis and antivenom administration. Here, we tried to produce highly specific antibodies in goose yolks for use in a paper-based microfluidic diagnostic kit, immunochromatographic test of viper (ICT-Viper), to distinguish RVs from other vipers and even cobra snakebite in Asia. We used indirect ELISA to monitor specific goose IgY production and western blotting to illustrate the interaction of avian or mammal antibody with venom proteins. The ICT-Viper was tested not only in prepared samples but also in stored patient serum to demonstrate its preliminary efficacy. The results revealed that specific anti-Daboia russelii IgY could be raised in goose eggs effectively without inducing adverse effects. When it was collocated with horse anti-Daboia siamensis antibody, which broadly reacted with most of the venom proteins of both types of Russell's viper, the false cross-reactivity was reduced, and the test showed good performance. The limit of detection was reduced to 10 ng/ml in vitro, and the test showed good detection ability in clinical snake envenoming case samples. The ICT-Viper performed well and could be combined with a cobra venom detection kit (ICT-Cobra) to create a multiple detection strip (ICT-VC), which broadens its applications while maintaining its detection ability for snake envenomation identification. Nonetheless, the use of the ICT-Viper in the South-East Asia region is pending additional laboratory and field investigations and regional collaboration. We believe that the development of this practical diagnostic tool marks the beginning of positive efforts to face the global snakebite issue.

Exploring prior diseases associated with incident late-onset Alzheimer's disease dementia.

Studies have identified prior conditions associated with late-onset Alzheimer's disease dementia (LOAD), but all prior diseases have rarely been screened simultaneously in the literature. Our objective in the present study was to identify prior conditions associated with LOAD and construct pathways for them. We conducted a population-based matched case-control study based on data collected in the National Health Insurance Research database of Taiwan and the Catastrophic Illness Certificate database for the years 1997-2013. Prior diseases definitions were based on the first three digits of the codes listed in the International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM). Inclusion criteria required that each ICD-code existed for at least 1 year and incurred at least 2 outpatient visits or inpatient diagnosis. The case group comprised 4,600 patients newly diagnosed with LOAD in 2007-2013. The LOAD patients were matched by sex and age to obtain 4,600 controls. Using stepwise multivariate logistic regression analysis, diseases were screened for 1, 2 …, 9 years prior to the first diagnosis of LOAD. Path analysis was used to construct pathways between prior diseases and LOAD. Our results revealed that the following conditions were positively associated with the incidence of LOAD: anxiety (ICD-code 300), functional digestive disorder (ICD code 564), psychopathology-specific symptoms (ICD-code 307), disorders of the vestibular system (ICD-code 386), concussion (ICD-code 850), disorders of the urethra and urinary tract (ICD-code 599), disorders of refraction and accommodation (ICD-code 367), and hearing loss (ICD-code 389). A number of the prior diseases have previously been described in the literature in a manner identical to that in the present study. Our study supports the assertion that mental, hearing, vestibular system, and functional digestive disorders may play an important role in the pathogenesis of LOAD.

Focal Loss for Dense Object Detection.

The highest accuracy object detectors to date are based on a two-stage approach popularized by R-CNN, where a classifier is applied to a sparse set of candidate object locations. In contrast, one-stage detectors that are applied over a regular, dense sampling of possible object locations have the potential to be faster and simpler, but have trailed the accuracy of two-stage detectors thus far. In this paper, we investigate why this is the case. We discover that the extreme foreground-background class imbalance encountered during training of dense detectors is the central cause. We propose to address this class imbalance by reshaping the standard cross entropy loss such that it down-weights the loss assigned to well-classified examples. Our novel Focal Loss focuses training on a sparse set of hard examples and prevents the vast number of easy negatives from overwhelming the detector during training. To evaluate the effectiveness of our loss, we design and train a simple dense detector we call RetinaNet. Our results show that when trained with the focal loss, RetinaNet is able to match the speed of previous one-stage detectors while surpassing the accuracy of all existing state-of-the-art two-stage detectors. Code is at: https://github.com/facebookresearch/Detectron.

Exploiting Signaling Pathways and Immune Targets Beyond the Standard of Care for Ewing Sarcoma.

Ewing sarcoma (ES) family of tumors includes bone and soft tissue tumors that are often characterized by a specific translocation between chromosome 11 and 22, resulting in the EWS-FLI1 fusion gene. With the advent of multi-modality treatment including cytotoxic chemotherapy, surgery, and radiation therapy, the prognosis for patients with ES has substantially improved. However, a therapeutic plateau is now reached for both localized and metastatic disease over the last two decades. Burdened by the toxicity limits associated with the current frontline systemic therapy, there is an urgent need for novel targeted therapeutic strategies. In this review, we discuss the current treatment paradigm of ES, and explore preclinical evidence and emerging treatments directed at tumor signaling pathways and immune targets.

Likelihood of a fecal occult blood test uptake among older adults: comparisons between health professionals and healthcare volunteers based on the health belief model.

BACKGROUND: Health professionals and healthcare volunteers play a critical role in promoting uptake of the fecal occult blood test (FOBT), which is an effective screening method for colorectal cancer. However, previous studies paid less attention to investigating both groups regarding their intention to undergo the test. This study used the Health Belief Model (HBM) to explore the likelihood of an FOBT uptake among health professionals and healthcare volunteers aged 50 years or older. METHODS: A cross-sectional survey was conducted at public health centers in a county in northern Taiwan. Health professionals and healthcare volunteers were invited to complete the questionnaires. Overall, 391 valid questionnaires were obtained (response rate = 93.10%). Structural equation modeling was used to examine the associations among the variables based on the HBM. RESULTS: The HBM explained 45, 44, and 50% of the variance in the likelihood of undergoing an FOBT in all participants, health professionals, and healthcare volunteers, respectively. The explained variance in healthcare volunteers outweighed that of professionals by 6%. Perceived benefits and self-efficacy significantly affected the likelihood of undergoing an FOBT. Self-efficacy significantly mediated the effects of perceived severity, benefits, and barriers on the likelihood of an FOBT uptake. A borderline significant difference in structural coefficients was found across groups. CONCLUSIONS: The HBM model was used to examine the likelihood of an FOBT uptake among health professionals and healthcare volunteers, and the results showed that self-efficacy was the optimal predictor of the likelihood of an FOBT uptake, followed by perceived benefits. Future multifactorial interventions to promote FOBT uptake among health professionals and healthcare volunteers aged 50-75 years could include these significant factors.

Effect of Seasonal Change on Testicular Protein Expression in White Roman Geese.

The purpose of this study was to investigate the change in protein expression in the testes of ganders at various breeding stages. A total of nine 3-year-old male White Roman ganders were used. The blood and testis samples were collected at the nonbreeding, sexual reactivation, and breeding stages for sex hormone analysis and proteomic analysis, respectively. The testicular weight and serum testosterone observed for ganders at the breeding stage were higher than those for ganders at nonbreeding and sexual reactivation stages (P < 0.05). There were 124 protein spots differentially expressed in the testes of ganders at various reproductive stages. A total of 107 protein spots of 74 proteins was identified through mass spectrometry. Most of the differentially expressed proteins were responsible for the molecular functions of protein binding (24%) and catalytic activity (16%). A functional pathway analysis suggested that proteins involved in steroidogenesis, metabolism, and spermatogenesis pathways changed in the White Roman geese at various reproductive stages. In conclusion, ganders at various reproductive stages exhibited different levels of testosterone and protein expression in the testes. The varied levels of the proteins might be essential and unique key factors in seasonal reproduction in ganders.

A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5.

BACKGROUND: An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition. METHOD: A Tmloop inserted CtCel5E and its related mutants were produced to investigate the role of Tmloop in catalysis. Crystal structure of CtCel5E-TmloopF267A followed by site-direct mutagenesis reveals the mechanism. RtCelB, a homolog with Tmloop was identified to have mannanase activity. RESULT: Tmloop incorporation enables CtCel5E to gain mannanase activity. Tyr270, His277, and Trp282 in the Tmloop are indispensable for CtCel5E-Tmloop catalysis, and weakening hydrophobic environment near the Tmloop enhances enzyme kcat. Using our newly identified loop motif to search for structurally conserved homologs in other subfamilies of GH5, we identified RtCelB. This homolog, originally annotated as a cellulase also possesses mannanase and xylanase activities. CONCLUSION: Our studies show that Tmloop enhances GH5 enzyme promiscuity and plays a role in catalysis. GENERAL SIGNIFICANCE: The study identified a loop of GH5 for mannan recognition and catalysis. Weakening the hydrophobic environment near the loop can also enhance the enzyme catalytic rate. Our findings provide a new insight on mannan recognition and activity enhancement of GH5.

Interrogation of side chain biases for oligomannose recognition by antibody 2G12 via structure-guided phage display libraries.

Monoclonal antibodies (mAbs) are essential reagents for deciphering gene or protein function and have been a fruitful source of therapeutic and diagnostic agents. However, developing anticarbohydrate antibodies to target glycans for those purposes has been less successful because the molecular basis for glycan-mAb interactions is poorly understood relative to protein- or peptide-binding mAbs. Here, we report our investigation on glycan-mAb interactions by using the unique architectural scaffold of 2G12, an antibody that targets oligomannoses on the HIV-1 glycoprotein gp120, as the template for engineering highly specific mAbs to target glycans. We first analyzed 24 different X-ray structures of antiglycan mAbs from the Protein Data Bank to determine side chain amino acid distributions in of glycan-mAb interactions. We identified Tyr, Arg, Asn, Ser, Asp, and His as the six most prevalent residues in the glycan-mAb contacts. We then utilized this information to construct two phage display libraries ("Lib1" and "Lib2") in which positions on the heavy chain variable domains of 2G12 were allowed to vary in restricted manner among Tyr, Asp, Ser, His, Asn, Thr, Ala and Pro to interrogate the minimal physicochemical requirements for oligomannose recognition. We analyzed the sequences of 39 variants from Lib1 and 14 variants from Lib2 following selection against gp120, the results showed that there is a high degree of malleability within the 2G12 for glycan recognitions. We further characterized five unique phage clones from both libraries that exhibited a gp120-specific binding profile. Expression of two of these variants as soluble mAbs indicated that, while specificity of gp120-binding was retained, the affinity of these mutants was significantly reduced relative to WT 2G12. Nonetheless, the results indicate these is some malleability in the identity of contact residues and provide a novel insight into the nature of glycan-antibody interactions and how they may differ from protein-antibody binding interactions.

Effects of monochromatic light sources on sex hormone levels in serum and on semen quality of ganders.

Light is an essential external factor influencing various physiological processes, including reproductive performance, in birds. Although several attempts have been made to understand the effect of light on poultry production, the effect of light of a particular wavelength (color) on the reproductive function in geese remains unclear. This study evaluated the effect of various monochromatic light sources on the levels of sex hormone and on semen quality of ganders. Of 30 male White Roman geese in their third reproductive season (average age=3 years), 27 were divided into three groups receiving monochromatic white or red or blue lights. The birds were kept in an environmentally controlled house with a lighting photoperiod of 7L:17D for six weeks as the adaptation period. The photoperiod was subsequently changed to 9L:15D and maintained for 24 weeks. Three ganders at the beginning of the study and three from each group at the end of the adjusting period and the 20th and 30th week of the study period were sacrificed, and their testes and blood samples were collected for determining the sex hormone levels. Semen samples were collected for determining semen quality parameters, including the semen collection index, sperm concentration, semen volume, sperm motility, sperm viability, sperm morphology, and semen quality factor. The results showed that the testosterone and estradiol levels remained unchanged in all three groups at all time points. The ratio of testosterone to estradiol of ganders exposed to white light was significantly higher than that of ganders exposed to red light at the 30th week (P<0.05). Semen collection index and sperm viability of ganders exposed to blue light were significantly the lowest (P<0.05). Moreover, sperm motility, sperm viability, and percentage of morphologically normal spermatozoa of ganders in white light were the highest (P<0.05). In conclusion, the results of this study suggested that artificial illumination with white light may maintain a better semen quality than that with red or blue lights in ganders.

A stabilization device that promotes the efficiency of cardiopulmonary resuscitation during ambulance transportation to the level as under non-moving conditions.

BACKGROUND: The survival rate of patients with out-of-hospital cardiac arrest is low, and measures to improve the quality of cardiopulmonary resuscitation (CPR) during ambulance transportation are desirable. We designed a stabilization device, and in a randomized crossover trial we found performing CPR in a moving ambulance with the device (MD) could achieve better efficiency than that without the device (MND), but the efficiency was lower than that in a non-moving ambulance (NM). PURPOSE: To evaluate whether a modified version of the stabilization device, can promote further the quality of CPR during ambulance transportation. METHODS: Participants of the previous study were recruited, and they performed CPR for 10 minutes in a moving ambulance with the modified version of the stabilization device (MVSD). The primary outcomes were effective chest compressions and no-flow fraction recorded by a skill-reporter manikin. The secondary outcomes included back pain, physiological parameters, and the participants' rating about the device after performing CPR. RESULTS: The overall effective compressions in 10 minutes were 86.4±17.5% for NM, 60.9±14.6% for MND, 69.7±22.4% for MD, and 86.6%±13.2% for MVSD (p<0.001). Whereas changes in back pain severity and physiology parameters were similar under all conditions, MVSD had the lowest no-flow fraction. Differences in effective compressions and the no-flow fraction between MVSD and NM did not reach statistical significance. CONCLUSIONS: The use of the modified device can improve quality of CPR in a moving ambulance to a level similar to that in a non-moving condition without increasing the severity of back pain.

A stabilization device to improve the quality of cardiopulmonary resuscitation during ambulance transportation: a randomized crossover trial.

BACKGROUND: The quality of cardiopulmonary resuscitation (CPR) during ambulance transportation is suboptimal, and therefore measures that can improve the quality are desirable. PURPOSE: To evaluate whether the use of a stabilization device can improve the quality of CPR during ambulance transportation. METHODS: This randomized controlled crossover trial enrolled 22 experienced ambulance officers. Each participant performed CPR in an ambulance under three conditions with 72 h apart, each condition for 10 min: non-moving (NM), moving without device (MND), and moving with device (MD). The sequences of conditions were randomized. The primary outcomes were effective chest compressions recorded by the Laerdal Resusci-Anne Skill-reporter manikin. The secondary outcomes included the severity of back pain scored using the Brief Pain Inventory short-form, the physiology parameter before and after CPR, and the changes in postural stability which was represented by the sway index (SI) of lower back measured using a goniometer. RESULTS: The overall effective compressions in 10 min were 87.0±17% for NM, 59.0±19% for MND, and 69.0±23% for MD (p<0.001). Compared to MND, MD had a lower no-flow fraction while driving on curved sections (0.04 vs. 0.29, p<0.001). Whereas the pain severity and social interference scores were similar under all conditions, MND had a higher SI than MD and NM. CONCLUSIONS: The use of a stabilization device can improve the quality of CPR and posture stability during ambulance transportation, although the effects on the severity of back pain were not significant.

Sequence, cloning, and analysis of the fluvirucin B1 polyketide synthase from Actinomadura vulgaris.

Fluvirucin B1 , produced by Actinomadura vulgaris, is a 14-membered macrolactam active against a variety of infectious fungi as well as influenza A. Despite considerable interest from the synthetic community, very little information is available regarding the biosynthetic origins of the fluvirucins. Herein, we report the identification and initial characterization of the fluvirucin B1 polyketide synthase and related enzymes. The cluster consists of five extender modules flanked by an N-terminal acyl carrier protein and C-terminal thioesterase domain. All but one of the synthase modules contain the full complement of tailoring domains (ketoreductase, dehydratase, and enoyl reductase) as determined by sequence homology with known polyketide synthases. Acitve site analyses of several key components of the cluster are performed to further verify that this gene cluster is associated with production of fluvirucin B1 . This work will both open doors toward a better understanding of macrolactam formation and provide an avenue to genetics-based diversification of fluvirucin structure.

Correlation between substituent constants and hyperpolarizabilities for di-substituted trans-azobenzenes.

Nonlinear optical properties of a series of disubstituted trans-azobenzenes were studied. The structures were fully optimized by B3LYP/6-31+G* and both static polarizabilities and hyperpolarizabilities were then calculated by the derivative method. In order to show the relationships between dipole moments, (hyper)polarizabilities and the structures, three kinds of substituent constants were applied to correlate with both ground state dipole moment and hyperpolarizabilities. Both physical properties have a satisfactory correlation with substituent constants Σσ(+/-) and bond length alternation. Overall, the electronic excitation contribution to the hyperpolarizabilities is rationalized in terms of the two-level model.

Brain-derived neurotrophic factor-transfected and nontransfected 3T3 fibroblasts enhance migratory neuroblasts and functional restoration in mice with intracerebral hemorrhage.

Neurogenesis via the activation of endogenous neural progenitor cells is a potential treatment strategy for brain injury, including intracerebral hemorrhage (ICH). We assessed the efficacy of combined cell and brain-derived neurotrophic factor (BDNF) treatment in a mouse model of ICH induced by intracerebral collagenase injection. Complementary DNAs of mouse BDNF were transfected into cell lines of 3T3 fibroblasts. The expression and bioactivity of BDNF were analyzed by immunocytochemistry, Western blot, ELISA, and functional assays. Hematoma area and brain tissue loss were assessed by magnetic resonance imaging. The BDNF-transfected or nontransfected 3T3 fibroblasts were implanted as a growth factor source in mice with ICH. Neurogenesis and functional recovery were evaluated 15 days after ICH. The BDNF-treated mice had the most doublecortin-positive cells near lesions and the least brain tissue loss in all groups. Both cell treatment groups had abundant newly proliferative glial fibrillary acidic protein-positive cells and better functional improvement than controls. These results indicate that fibroblast transplantation, together with recombinant BDNF treatment, after ICH is beneficial in mice. The early functional recovery may result from the growth factors that are provided or evoked by the implanted grafts. These results suggest a potential approach for combining gene and cell therapy for ICH treatment.

A mechanism-based fluorescence transfer assay for examining ketosynthase selectivity.

Since their discovery, polyketide synthases have received massive attention from researchers hoping to harness their potential as a platform for generating new and improved therapeutics. Despite significant strides toward this end, inherent specificities within the enzymes responsible for polyketide production have severely limited these efforts. We have developed a mechanism-based, fluorescence transfer assay for a key enzyme component of all polyketide synthases, the ketosynthase domain. As demonstrated, this method can be used with both ketosynthase-containing didomains and full modules. As proof of principle, the ketosynthase domain from module 6 of the 6-deoxyerythronolide synthase is examined for its ability to accept a variety of simple thioester substrates. Consistent with its natural hexaketide substrate, we find that this ketosynthase prefers longer, α-branched thioesters and its ability to distinguish these structural features is quite remarkable. Substrate electronics are also tested via a variety of p-substituted aromatic groups. In all, we expect this technique to find considerable use in the field of polyketide biosynthesis and engineering due to its extraordinary simplicity and very distinct visible readout.