Development of a humanized anti-FABP4 monoclonal antibody for potential treatment of breast cancer.

BACKGROUND: Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus potentially offering a new target for breast cancer treatment. METHODS: We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. RESULTS: Herein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. CONCLUSIONS: Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases.

Cytological evidence of BSD2 functioning in both chloroplast division and dimorphic chloroplast formation in maize leaves.

BACKGROUND: Maize bsd2 (bundle sheath defective2) is a classical C4 mutant with defective C4 photosynthesis, accompanied with reduced accumulation of Rubisco (ribulose bisphosphate carboxylase oxygenase) and aberrant mature chloroplast morphology in the bundle sheath (BS) cells. However, as a hypothetical chloroplast chaperone, the effects of BSD2 on C4 chloroplast development have not been fully examined yet, which precludes a full appreciation of BSD2 function in C4 photosynthesis. The aims of our study are to find out the role ofBSD2 in regulating chloroplasts development in maize leaves, and to add new insights into our understanding of C4 biology. RESULTS: We found that at the chloroplast maturation stage, the thylakoid membranes of chloroplasts in the BS and mesophyll (M) cells became significantly looser, and the granaof chloroplasts in the M cells became thinner stacking in the bsd2 mutant when compared with the wildtype plant. Moreover, at the early chloroplast development stage, the number of dividing chloroplasts and the chloroplast division rate are both reduced in the bsd2 mutant, compared with wild type. Quantitative reverse transcriptase-PCR analysis revealed that the expression of both thylakoid formation-related genesand chloroplast division-related genes is significantly reduced in the bsd2 mutants. Further, we showed that BSD2 interacts physically with the large submit of Rubisco (LS) in Bimolecular Fluorescence Complementation assay. CONCLUSIONS: Our combined results suggest that BSD2 plays an essential role in regulating the division and differentiation of the dimorphic BS and M chloroplasts, and that it acts at a post-transcriptional level to regulate LS stability or assembly of Rubisco.

Determination of the FABP5 expression profile in skin lesions of an IMQ-induced psoriasis mouse model using flow cytometry.

The fatty acid-binding protein 5 (FABP5) is a key player in psoriasis development. Therefore, characterizing the expression profile of FABP5 in various cell types within both layers of psoriatic skin is important. Here, we present a protocol that describes steps for an imiquimod-induced psoriasis mouse model and preparation of epidermal and dermal single-cell suspensions. We then detail procedures to detect the FABP5 expression profile in skin keratinocytes and immune cells using intracellular flow cytometry staining. For complete details on the use and execution of this protocol, please refer to Hao et al.1.

Surface Modification of Silicon Nanowires with Siloxane Molecules for High-Performance Hydrovoltaic Devices.

Hydrovoltaic devices (HDs) based on silicon nanowires (SiNWs) have attracted significant attention due to their potential of high output power and good compatibility with Si-based photovoltaic devices for integrated power systems. However, it remains a major challenge to further improve the output performance of SiNW HDs for practical applications. Here, a new strategy to modify the surface of SiNWs with siloxane molecules is proposed to improve the output performance of the SiNW HDs. After modification, both the open-circuit voltage (Voc) and short-circuit current density (Jsc) of n-type SiNW HDs can be improved by approximately 30%, while the output power density can be greatly increased by over 200%. With siloxane modification, Si-OH groups on the surface of typical SiNWs are replaced by Si-O-Si chemical bonds that have a weaker electron-withdrawing capability. More free electrons in n-type SiNWs are liberated from surface bound states and participate in directed flow induced by water evaporation, thereby improving the output performance of HDs. The improved performance is significant for system integration applications as it reduces the number of required devices. Three siloxane-modified SiNW HDs in series are able to drive a 2 V light-emitting diode (LED), whereas four unmodified devices in series are initially needed for the same task. This work provides a simple yet effective strategy for surface modification to improve the output performance of SiNW HDs. Further research into the effect of different surface modifications on the performance of SiNW HDs will greatly promote their performance enhancement and practical applications.

FABP4-mediated lipid accumulation and lipolysis in tumor associated macrophages promote breast cancer metastasis.

A high density of tumor-associated macrophages (TAMs) is associated with poorer prognosis and survival in breast cancer patients. Recent studies have shown that lipid accumulation in TAMs can promote tumor growth and metastasis in various models. However, the specific molecular mechanisms that drive lipid accumulation and tumor progression in TAMs remain largely unknown. Herein, we demonstrated that unsaturated fatty acids (FAs), unlike saturated ones, are more likely to form lipid droplets in macrophages. Specifically, unsaturated FAs, including linoleic acids (LA), activate the FABP4/CEBPα pathway, leading to triglyceride synthesis and lipid droplet formation. Furthermore, FABP4 enhances lipolysis and FA utilization by breast cancer cells, which promotes cancer cell migration in vitro and metastasis in vivo . Notably, a deficiency of FABP4 in macrophages significantly reduces LA-induced lipid metabolism. Therefore, our findings suggest FABP4 as a crucial lipid messenger that facilitates unsaturated FA-mediated lipid accumulation and lipolysis in TAMs, thus contributing to the metastasis of breast cancer. Highlights: Unlike saturated fatty acids, unsaturated fatty acids preferentially promote lipid droplet formation in macrophages.Unsaturated fatty acids activate the FABP4/CEBPα axis for neutral lipid biosynthesis in macrophagesDeficiency of FABP4 compromised unsaturated fatty acid-mediated lipid accumulation and utilization in macrophagesFABP4-mediated lipid metabolism in macrophages contributes to breast cancer metastasis.

Gibberellin indirectly promotes chloroplast biogenesis as a means to maintain the chloroplast population of expanded cells.

Chloroplast biogenesis needs to be well coordinated with cell division and cell expansion during plant growth and development to achieve optimal photosynthesis rates. Previous studies showed that gibberellins (GAs) regulate many important plant developmental processes, including cell division and cell expansion. However, the relationship between chloroplast biogenesis with cell division and cell expansion, and how GA coordinately regulates these processes, remains poorly understood. In this study, we showed that chloroplast division was significantly reduced in the GA-deficient mutants of Arabidopsis (ga1-3) and Oryza sativa (d18-AD), accompanied by the reduced expression of several chloroplast division-related genes. However, the chloroplasts of both mutants exhibited increased grana stacking compared with their respective wild-type plants, suggesting that there might be a compensation mechanism linking chloroplast division and grana stacking. A time-course analysis showed that cell expansion-related genes tended to be upregulated earlier and more significantly than the genes related to chloroplast division and cell division in GA-treated ga1-3 leaves, suggesting the possibility that GA may promote chloroplast division indirectly through impacting leaf mesophyll cell expansion. Furthermore, our cellular and molecular analysis of the GA-response signaling mutants suggest that RGA and GAI are the major repressors regulating GA-induced chloroplast division, but other DELLA proteins (RGL1, RGL2 and RGL3) also play a role in repressing chloroplast division in Arabidopsis. Taken together, our data show that GA plays a critical role in controlling and coordinating cell division, cell expansion and chloroplast biogenesis through influencing the DELLA protein family in both dicot and monocot plant species.

Establishing diagnostic features for identifying the mucosa and submucosa of normal and cancerous gastric tissues by multiphoton microscopy.

BACKGROUND: Establishing diagnostic features is essential and significant for developing multiphoton endoscopy to make an early diagnosis of gastric cancer at the cellular level. Until now, these diagnostic features have not been clearly described and understood. DESIGN: Study of diagnostic features based on multiphoton microscopy (MPM). OBJECTIVE: Establishing diagnostic features to identify the mucosa and submucosa of human normal and cancerous gastric tissues by investigating their multiphoton microscopic images. SETTING: Fujian Normal University and Fujian Provincial Tumor Hospital. PATIENTS: Ten pairs of normal and cancerous specimens were obtained from 10 patients (ages 51-68 years) undergoing radical gastrectomy. INTERVENTIONS: MPM was performed on specimens. MAIN OUTCOME MEASUREMENTS: Establishment of diagnostic features. RESULTS: MPM has the ability to exhibit not only the mucosal and submucosal microstructures of normal and cancerous gastric tissues but also the distribution and content of abnormal cells in these 2 layers. More importantly, it can provide the diagnostic features to qualitatively and quantitatively differentiate between normal and cancerous gastric tissues. LIMITATIONS: The selection bias and preparation of specimen. CONCLUSIONS: These findings provide the groundwork for further establishing diagnostic criteria.

Characteristics of scar margin dynamic with time based on multiphoton microscopy.

Scar margins dynamic with time were quantitatively characterized using multiphoton microscopy (MPM). 2D large-area and 3D focused images of elastin and collagen at scar margins were obtained to extract quantitative parameters. An obvious boundary was observed at the scar margin, showing altered morphological patterns of elastin and collagen on both sides. Content alteration of elastin and collagen between the two sides of boundary were defined to characterize scar margins from different individuals. The statistical results from 15 normal scar samples strongly demonstrated that content alteration degree of elastin and collagen had decreasing tendency with the increase of patient age or scar duration, consistent with the fact of normal scars regressing spontaneously over time. It indicated that alteration degree can potentially serve as quantitative indicators to examine wound healing and scar progression over time. With the advent of clinical portable multiphoton endoscopes, the MPM technique can be applied in tracking scar formation and progression in vivo by examination of scar margin.

Stromal optical properties: differentiating normal and cancerous stroma.

This work reports on the measurement of optical properties from nine normal and cancerous human esophageal stroma pairs using reflectance-based confocal microscopy. It was found that the scattering coefficient of cancerous stroma is significantly lower than that of normal stroma. The results suggest that the decreased scattering in cancerous stroma may provide a possible indicator for differentiating normal and cancerous stroma.

Extracting diagnostic stromal organization features based on intrinsic two-photon excited fluorescence and second-harmonic generation signals.

Intrinsic two-photon excited fluorescence (TPEF) and second-harmonic generation (SHG) signals are shown to differentiate between normal and neoplastic human esophageal stroma. It was found that TPEF and SHG signals from normal and neoplastic stroma exhibit different organization features, providing quantitative information about the biomorphology and biochemistry of tissue. By comparing normal with neoplastic stroma, there were significant differences in collagen-related changes, elastin-related changes, and alteration in proportions of matrix molecules, giving insight into the stromal changes associated with cancer progression and providing substantial potential to be applied in vivo to the clinical diagnosis of epithelial precancers and cancers.

Jadassohn-Pellizzari anetoderma: study of multiphoton microscopy based on two-photon excited fluorescence and second harmonic generation.

Anetoderma is a rare skin disease with loss of dermal elastic tissue resulting in clinically localized areas of flaccid or herniated sack-like skin. In this study, we report a case of Jadassohn-Pellizzari anetoderma, in a 21-year-old Chinese female with an 18-year history of progressively generalized wrinkled skin lesions. Multiphoton microscopy based on two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) was firstly employed to investigate the pathological process from unaffected skin to the erythematous phase and finally with affected skin of this case. The results showed that the normal elastic fibers in unaffected skin were almost completely absent in erythematous skin tissue, then replaced by a lot of elastic fibers with granular morphology in affected skin, which was consistent with the histopathological results. The obvious changes in collagen fibers and the occurrence of inflammatory cell infiltration in erythematous tissue suggested that the variations of these two components were also the main pathogenesis of anetoderma, except for the deficiency of elastic fibers. Based on these data, we demonstrated that multiphoton microscopy was a promising tool for non-invasive investigation of the pathology of anetoderma at nearly histological resolution, and has potential for observing the dermatological dynamic processes for living specimens because it is based on the intrinsic signals of tissue components.

Multiphoton laser scanning microscopy of localized scleroderma.

BACKGROUND/PURPOSE: A real-time, non-invasive method will confer a benefit for the diagnosis and treatment of localized scleroderma (LS) in the clinic. The aim of this work was to demonstrate the potential of multiphoton laser scanning microscopy (MPLSM) for diagnosing LS and monitoring the treatment response in vivo. METHODS: Three sclerodermatous skin specimens and two normal skin specimens were investigated using MPLSM based on two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). MPLSM consists of a femtosecond Ti:sapphire laser and a scanning inverted microscope. Several parameters such as the epidermal thickness, the orientation ratio index of collagen bundles (ORICB), the spacing of collagen fibrin as well as the SHG to TPEF index of the dermis (STID) were developed to quantitatively discriminate the sclerodermatous skin from the normal skin. RESULTS: The morphological differences were visualized obviously in the TPEF/SHG images of human skin (normal and sclerodermatous). The values of the developed parameters in normal skin were significantly different from that in sclerodermatous skin (P<0.05). CONCLUSION: MPLSM could discriminate the sclerodermatous skin from the normal skin. With the advent of the clinical portability of typical MPLSM, this technique has great potential for application in the in vivo diagnosis of LS as well as for monitoring the treatment response.

Two-layered multiphoton microscopic imaging of cervical tissue.

We demonstrate the application of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second harmonic generation signals in imaging two-layered structures of cervical tissue in mice. It was found that MPM is effective for visualizing two-layered cervical microstructures, monitoring the metabolic activity and the density (nuclear-to-cytoplasmic) of epithelial cells, measuring the size of epithelial cell nuclei and the thickness of the epithelial layer, and quantifying collagen in the stroma. These results suggest that, with the advent of clinical portability of typical multiphoton endoscopes, the MPM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of cervical cancer.

Nonlinear optical microscopy of the bronchus.

Because of frequent exposure to carcinogens, the bronchus is prone to early pathologic alterations. The assessment of these early changes is of key significance in physiological studies and disease diagnosis of the bronchus. We utilize nonlinear optical microscopy (NLOM) to image mouse bronchial tissue based on intrinsic nonlinear optical contrast. Our results show that NLOM is effective for imaging the bronchial intact microstructural components, providing quantitative information about the biomorphology and biochemistry of tissue. Our findings also display that NLOM can provide a two-photon ratiometric redox fluorometry, based on mitochondrial signals and reduced pyridine nucleotide (NADH and NADPH) and oxidized flavoproteins (Fp) signals, to assess the metabolic state of the epithelial cells and chondrocytes. It was found that NLOM can offer a sensitive tool, based on the second-harmonic signal depth-dependent decay, to obtain quantitative information on the optical property of the stroma associated with normal and diseased tissue states. Our results suggest that with the advent of the clinical portability of typical nonlinear optical endoscopy, the NLOM technique has the potential to be applied in vivo to the clinical diagnosis and monitoring of bronchial disease.

Imaging collagen remodeling and sensing transplanted autologous fibroblast metabolism in mouse dermis using multimode nonlinear optical imaging.

Collagen remodeling and transplanted autologous fibroblast metabolic states in mouse dermis after cellular injection are investigated using multimode nonlinear optical imaging. Our findings show that the technique can image the progress of collagen remodeling in mouse dermis. It can also image transplanted autologous fibroblasts in their collagen matrix environment in the dermis, because of metabolic activity. It was also found that the approach can provide two-photon ratiometric redox fluorometry based on autologous fibroblast fluorescence from reduced nicotinamide adenine dinucleotide coenzyme and oxidized flavoproteins for sensing the autologous fibroblast metabolic state. These results show that the multimode nonlinear optical imaging technique may have potential in a clinical setting as an in vivo diagnostic and monitoring system for cellular therapy in plastic surgery.

Multiphoton microscopic imaging of adipose tissue based on second-harmonic generation and two-photon excited fluorescence.

The fresh adipose tissue was investigated by the use of multiphoton microscopy (MPM) based on two-photon excited fluorescence and second-harmonic generation (SHG). Microstructure of collagen and adipose cells in the adipose tissue is clearly imaged at a subcellular level with the excitation light wavelengths of 850 and 730 nm, respectively. The emission spectrum of collagen SHG signal and NADH and FAD fluorescence signal can also be obtained, which can be used to quantify the content of collagen and adipose cells and reflect the degree of pathological changes when comparing normal tissue with abnormal adipose tissue in the same condition. The results indicate that MPM has the potential to be applied to investigate the adipose tissue and can be used in the research field of lipid and connective tissues.

The layered-resolved microstructure and spectroscopy of mouse oral mucosa using multiphoton microscopy.

The layered-resolved microstructure and spectroscopy of mouse oral mucosa are obtained using a combination of multiphoton imaging and spectral analysis with different excitation wavelengths. In the keratinizing layer, the keratinocytes microstructure can be characterized and the keratinizing thickness can be measured. The keratin fluorescence signal can be further characterized by emission maxima at 510 nm. In the epithelium, the cellular microstructure can be quantitatively visualized with depth and the epithelium thickness can be determined by multiphoton imaging excited at 730 nm. The study also shows that the epithelial spectra excited at 810 nm, showing a combination of NADH and FAD fluorescence, can be used for the estimation of the metabolic state in epithelium. Interestingly, a second-harmonic generation (SHG) signal from DNA was observed for the first time within the epithelial layer in backscattering geometry and provides the possibility of analyzing the chromatin structure. In the stroma, the combination of multiphoton imaging and spectral analysis excited at 850 nm in tandem can obtain quantitative information regarding the biomorphology and biochemistry of stroma. Specifically, the microstructure of collagen, minor salivary glands and elastic fibers, and the optical property of the stroma can be quantitatively displayed. Overall, these results suggest that the combination of multiphoton imaging and spectral analysis with different excitation wavelengths has the potential to provide important and comprehensive information for early diagnosis of oral cancer.

Sequential multitrack nonlinear ex vivo imaging of esophageal stroma based on backscattered second-harmonic generation and two-photon autofluorescence.

We demonstrate the technique of subsequent multitrack nonlinear imaging based on backscattered second-harmonic generation (B-SHG) and two-photon autofluorescence (TPA) to obtain large-area, high-contrast, submicron-resolution image ex vivo of esophageal stroma. Our findings show that this technique is effective in improving the B-SHG/TPA image contrast. It was found that the method can quantitatively obtain microscopic structural and biochemical information on stroma. Our work suggests that the technique has the potential to provide accurate and comprehensive information in determining the physiological and pathological states of the esophagus.

Spectral characteristics of autofluorescence and second harmonic generation from ex vivo human skin induced by femtosecond laser and visible lasers.

The spectral properties of one-photon, two-photon excited autofluorescence and second harmonic generation (SHG) from ex vivo human skin induced by a femtosecond (fs) laser and three visible lasers in backscattering geometry are systematically investigated. Our experimental results indicate that peak position of autofluorescence spectra from the dermis and epidermis shift toward long wavelengths, and the fluorescent intensity decreases when the excitation wavelength increases due to an effect of the excitation wavelength on autofluorescence signals. However, the intensity of the SHG signal in collagen has the maximal value of 800 nm excitation wavelength. This may be the result that the energy of the SHG signal is in resonance with an electronic absorption band. The two-photon excited autofluorescence and SHG intensity all obey a quadratical dependence on the excitation power. Compared with the two-photon excited fluorescence and SHG, the one-photon excited fluorescence in the dermis and epidermis exhibits different spectral characteristics. The investigation of the spectral characteristics of autofluorescence and SHG from ex vivo human skin can provide new insights into morphologic structures and biochemical components of tissues, which are vital for improving the application of laser-induced autofluorescence and SHG spectroscopy technique for noninvasive in vivo tissue diagnostics.

Multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation.

Mouse is an important animal model to investigate skin physiological and pathological states. In this article, multiphoton microscopic imaging of in vivo hair mouse skin based on two-photon excited fluorescence and second harmonic generation was examined. Our results show that multiphoton microscopy can clearly display microstructure of stratum corneum, stratum spinosum, and dermis of in vivo mouse skin. The main components of epidermis and dermis such as corneocytes, spinosum cell, collagen fibers, and hair follicles can be distinctly identified in MPM images. Using the optional HRZ 200 fine focusing stage, thickness of different layers can be easily assessed. The results demonstrate that MPM can be regarded as an efficient method for in vivo investigation of skin physiological and pathological states by using hair mouse animal model.