Genetically encoded calcium indicators for visualizing dynamic cellular activity have greatly expanded our understanding of the brain. However, due to light scattering properties of the brain as well as the size and rigidity of traditional imaging technology, in vivo calcium imaging has been limited to superficial brain structures during head fixed behavioral tasks. This limitation can now be circumvented by utilizing miniature, integrated microscopes in conjunction with an implantable microendoscopic lens to guide light into and out of the brain, thus permitting optical access to deep brain (or superficial) neural ensembles during naturalistic behaviors. Here, we describe procedural steps to conduct such imaging studies using mice. However, we anticipate the protocol can be easily adapted for use in other small vertebrates. Successful completion of this protocol will permit cellular imaging of neuronal activity and the generation of data sets with sufficient statistical power to correlate neural activity with stimulus presentation, physiological state, and other aspects of complex behavioral tasks. This protocol takes 6–11 weeks to complete.

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily a1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a a1,6-fucosyltransferase. FUT8À/À CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8 À/À cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8 À/À CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8 À/À cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFNderived FUT8 À/À cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8 À/À cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8 À/À cell lines was fourfold higher than in the best-producing clones of FUT8 À/À cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8À phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies.

SUMMARY In vivo calcium imaging using a 1-photon-based miniscope and a microendoscopic lens enables studies of neural activities in freely behaving animals. However, the high and fluctuating background, the inevitable movements and distortions of imaging field, and the extensive spatial overlaps of fluorescent signals emitted from imaged neurons inherent in this 1-photon imaging method present major challenges for extracting neuronal signals reliably and automatically from the raw imaging data. Here, we develop a unifying algorithm called the miniscope 1-photon imaging pipeline (MIN1PIPE), which contains several stand-alone modules and can handle a wide range of imaging conditions and qualities with minimal parameter tuning and automatically and accurately isolate spatially localized neural signals. We have quantitatively compared MIN1PIPE with other existing partial methods using both synthetic and real datasets obtained from different animal models and show that MIN1PIPE has superior efficiency and precision in analyzing noisy miniscope calcium imaging data.

SUMMARY 19In vivo calcium imaging using 1-photon based miniscope and microendoscopic lens 20 enables studies of neural activities in freely behaving animals. However, the high and analyzing noisy miniscope calcium imaging data.

Androgen deprivation therapy (ADT), including enzalutamide, induces resistance in prostate cancer; ADT resistance is associated with neuroendocrine differentiation (NED) and tumor-associated macrophages (TAM). This study aimed to investigate the association between enzalutamide-induced NED and TAMs and its mechanism. The association between enzalutamide-induced NED and TAMs was investigated by IHC using prostate cancer tissues, enzalutamide-resistant mouse xenografts, and a coculture system. The underlying mechanisms were assessed using cytokine antibody arrays, ELISAs, chromatin immunoprecipitation, and other methods. An orthotopic prostate cancer mouse model was established to evaluate the effects of combined IL6 receptor (IL6R) and high mobility group box 1 (HMGB1) inhibition on enzalutamide resistance. High CD163 expression was observed in ADT-treated prostate cancer or castration-resistant prostate cancer (CRPC) tissues with high levels of neuron-specific enolase (NSE) and chromogranin A (CHGA) and in enzalutamide-resistant xenografts, indicating the crucial roles of NED and TAMs in enzalutamide resistance. Specifically, enzalutamide-induced HMGB1 expression facilitated TAM recruitment and polarization and drove NED via β-catenin stabilization. HMGB1-activated TAMs secreted IL6 to augment enzalutamide-induced NED and directly promote HMGB1 transcription via STAT3. Finally, inhibition of the IL6/STAT3 pathway by tocilizumab combined with HMGB1 knockdown inhibited enzalutamide-induced resistance in an orthotopic prostate cancer mouse model. Enzalutamide elevates HMGB1 levels, which recruits and activates TAMs. Moreover, IL6 secreted by HMGB1-activated TAMs facilitates the enzalutamide-induced NED of prostate cancer, forming a positive feedback loop between NED in prostate cancer and TAMs. The combined inhibition of IL6R and HMGB1 may serve as a new treatment for enzalutamide resistance in patients with advanced or metastatic prostate cancer. .

Cancer stem-like cells (CSC) contribute to the progression and androgen deprivation therapy (ADT) resistance of prostate cancer. As CSCs depend on their specific niche, including tumor-associated macrophages (TAM), elucidating the network between CSCs and TAMs may help to effectively inhibit the progression and ADT resistance of prostate cancer. The underlying intracellular mechanism that sustains the stem-like characteristics of CSCs in prostate cancer was assessed via RNA sequencing, co-immunoprecipitation, chromatin immunoprecipitation, and other assays. A coculture system and cytokine antibody arrays were used to examine the interaction network between CSCs and TAMs. In addition, an orthotopic prostate cancer model was established to evaluate the effects of the combined targeting of CSCs and their interaction with TAMs on ADT resistance. Autophagy-related gene 7 (ATG7) facilitated the transcription of OCT4 via β-catenin, which binds to the OCT4 promoter, promoting CSC characteristics in prostate cancer, including self-renewal, tumor initiation, and drug resistance. In addition, CSCs remodeled their specific niche by educating monocytes/macrophages toward TAMs, and the CSC-educated TAMs reciprocally promoted the stem-like properties of CSCs, progression and ADT resistance of prostate cancer via IL6/STAT3. Furthermore, the combined targeting of CSCs and their interaction with TAMs by inhibiting ATG7/OCT4 and IL6 receptor effectively ameliorated ADT resistance in an orthotopic prostate cancer model. Targeting CSCs and their niche may prove to be a more powerful strategy than targeting CSCs alone, providing a rational approach to ameliorating ADT resistance in prostate cancer. .

Ongoing changes in arousal influence sensory processing and behavioral performance. Yet the circuit-level correlates for this influence remain poorly understood. Here, we investigate how functional interaction between posterior parietal cortex (PPC) and lateral posterior (LP)/Pulvinar is influenced by ongoing fluctuations in pupil-linked arousal, which is a non-invasive measure of neuromodulatory tone in the brain. We find that fluctuations in pupil-linked arousal correlate with changes to PPC to LP/Pulvinar oscillatory interaction, with cortical alpha oscillations driving activity during low arousal states, and LP/Pulvinar driving PPC in the theta frequency band during higher arousal states. Active visual exploration by saccadic eye movements elicits similar transitions in thalamo-cortical interaction. Furthermore, the presentation of naturalistic video stimuli induces thalamo-cortical network states closely resembling epochs of high arousal in the absence of visual input. Thus, neuromodulators may play a role in dynamically sculpting the patterns of thalamo-cortical functional interaction that underlie visual processing.

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