Deep learning-assisted smartphone-based quantitative microscopy for label-free peripheral blood smear analysis.

Hematologists evaluate alterations in blood cell enumeration and morphology to confirm peripheral blood smear findings through manual microscopic examination. However, routine peripheral blood smear analysis is both time-consuming and labor-intensive. Here, we propose using smartphone-based autofluorescence microscopy (Smart-AM) for imaging label-free blood smears at subcellular resolution with automatic hematological analysis. Smart-AM enables rapid and label-free visualization of morphological features of normal and abnormal blood cells (including leukocytes, erythrocytes, and thrombocytes). Moreover, assisted with deep-learning algorithms, this technique can automatically detect and classify different leukocytes with high accuracy, and transform the autofluorescence images into virtual Giemsa-stained images which show clear cellular features. The proposed technique is portable, cost-effective, and user-friendly, making it significant for broad point-of-care applications.

Tissue-specific metabolomic profiling after cardiopulmonary bypass in fetal sheep.

Objective: Fetal cardiopulmonary bypass (CPB) is essential to fetal heart surgery, while its development is limited by vital organ dysfunction after CPB. Studying organ metabolism may help to solve this problem. The objective of this study was to describe the tissue-specific metabolic fingerprints of fetal sheep under CPB and to associate them with organ functions. Methods: Ten pregnant ewes at 90-120 days of gestation were randomly divided into two groups. The bypass group underwent a 1-h fetal CPB, whereas the control group underwent only a fetal sternotomy. During bypass, echocardiography, blood gases, and blood biochemistry were measured. After bypass, lambs were sacrificed, and tissues of the heart, liver, brain, kidney, and placenta were harvested. The metabolites extracted from these tissues were analyzed using non-targeted metabolomics based on liquid chromatography-mass spectrometry techniques. Results: All tissues except the placenta displayed significant metabolic changes, and the fetal heart displayed obvious functional changes. Fetal sheep that underwent CPB had common and tissue-specific metabolic signatures. These changes can be attributed to dysregulated lipid metabolism, altered amino acid metabolism, and the accumulation of plasticizer metabolism. Conclusion: Fetal CPB causes tissue-specific metabolic changes in fetal sheep. Studying these metabolic changes, especially cardiac metabolism, is of great significance for the study of fetal CPB.

Low-cost high-resolution photoacoustic microscopy of blood oxygenation with two laser diodes.

Optical-resolution photoacoustic microscopy (OR-PAM) has been widely used for imaging blood vessel and oxygen saturation of hemoglobin (sO2), providing high-resolution functional images of living animals in vivo. However, most of them require one or multiple bulky and costly pulsed lasers, hindering their applicability in preclinical and clinical settings. In this paper, we demonstrate a reflection-mode low-cost high-resolution OR-PAM system by using two cost-effective and compact laser diodes (LDs), achieving microvasculature and sO2 imaging with a high lateral resolution of ∼6 µm. The cost of the excitation sources has dramatically reduced by ∼20-40 times compared to that of the pulsed lasers used in state-of-the-art OR-PAM systems. A blood phantom study was performed to show a determination coefficient R 2 of 0.96 in linear regression analysis. Experimental results of in vivo mouse ear imaging show that the proposed dual-wavelength LD-based PAM system can provide high-resolution functional images at a low cost.

TMAO-Activated Hepatocyte-Derived Exosomes Are Widely Distributed in Mice with Different Patterns and Promote Vascular Inflammation.

BACKGROUND: Trimethylamine-N-oxide (TMAO) has been shown to be an important player in cardiovascular disease (CVD) by promoting vascular inflammation and endothelial dysfunction. We recently found that exosomes (Exos) released from TMAO-activated hepatocytes (TMAO-Exos) could significantly induce inflammation and endothelial dysfunction. However, understandings of how are the Exos secreted by hepatocytes, where are they distributed in vivo, and what effects will they have on vascular inflammation remain limited. The present study aimed to explore the hub genes involved in the production of TMAO-Exos and their distributions in vivo and effects on inflammation. METHODS: The transcriptome profiles of primary rat hepatocytes stimulated with TMAO were obtained from the GSE135856 dataset in the Gene Expression Omnibus repository, and the hub genes associated with Exos were screened and verified by qPCR. Next, Exos derived from TMAO-treated hepatocytes were isolated using differential centrifugation and given intravenously to mice. After 24 h, the distributions of DiI-labelled Exos were visualized with a fluorescence microscope, and the levels of proinflammatory genes in the aorta were detected by qPCR. RESULTS: Phgdh, Mdh2, Echs1, Rap2a, Gpd1l, and Slc3a2 were identified as hub genes that may be involved in the production of TMAO-Exos. And TMAO-Exos were found to be efficiently taken up by cardiomyocytes, hepatocytes, and endothelial cells in the aorta and gastrocnemius muscle. Furthermore, TMAO-Exos, but not control-Exos, could significantly promote the mRNA expressions of Tnf, Icam1, Sele, and Cox-2 in the aorta. CONCLUSIONS: We provided clues about how TMAO may stimulate hepatocytes to produce Exos and further offered evidence that Exos secreted by TMAO-treated hepatocytes could be widely distributed in vivo and promote vascular inflammation.

Ultraviolet photoacoustic microscopy with tissue clearing for high-contrast histological imaging.

Ultraviolet photoacoustic microscopy (UV-PAM) has been investigated to provide label-free and registration-free volumetric histological images for whole organs, offering new insights into complex biological organs. However, because of the high UV absorption of lipids and pigments in tissue, UV-PAM suffers from low image contrast and shallow image depth, hindering its capability for revealing various microstructures in organs. To improve the UV-PAM imaging contrast and imaging depth, here we propose to implement a state-of-the-art optical clearing technique, CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis), to wash out the lipids and pigments from tissues. Our results show that the UV-PAM imaging contrast and quality can be significantly improved after tissue clearing. With the cleared tissue, multilayers of cell nuclei can also be extracted from time-resolved PA signals. Tissue clearing-enhanced UV-PAM can provide fine details for organ imaging.

Central and Peripheral Cannulation for Cardiopulmonary Bypass in Fetal Sheep: A Comparative Study.

Objective: In-utero correction is an option for treatment of critical congenital heart diseases (CHDs). Fetal cardiac surgery for CHDs is dependent on the reliable use of fetal cardiopulmonary bypass (CPB), but this technology remains experimental. In this study, we established fetal CPB models with central and peripheral cannulation to explore the differences between the two cannulation strategies. Methods: Ten fetal sheep with 90-110 gestational days were randomized into central cannulation (n = 5) and peripheral cannulation (n = 5) groups. All fetal CPB models were successfully established. At each time point (0, 30, and 60 min after initiation of CPB), echocardiography was performed. Blood samples were also collected for blood gas analysis and tests of myocardial enzymes and liver and kidney function. Results: In the central cannulation group, right ventricular Tei index significantly increased (p = 0.016) over time. Compared with the peripheral cannulation group, the left ventricular Tei index of the central cannulation group was significantly higher (1.96 ± 0.31 vs. 0.45 ± 0.19, respectively; p = 0.028) and the stroke volume was lower (0.46 ± 0.55 vs. 2.13 ± 0.05, respectively; p = 0.008) at 60 min after CPB. Levels of liver and kidney injury markers and of acid-base balance, including alanine aminotransferase (ALT), aspartate aminotransferase/ALT ratio, blood urea nitrogen (BUN), BUN/creatinine ratio, base excess and bicarbonates, were significantly higher for peripheral than for central cannulation. Other important physiologic parameters, including heart rate, blood pressure, myocardial enzymes, umbilical artery beat index and resistance index, left ventricular Tei index, and left and right ventricular stroke volume, were comparable between the two groups. Conclusions: Both central and peripheral cannulations can be used to establish fetal CPB models. Central cannulation causes more adverse impacts for cardiac function, whereas peripheral cannulation is more susceptible to complications related to inadequate organ perfusion.

Regulating Surface and Grain-Boundary Structures of Ni-Rich Layered Cathodes for Ultrahigh Cycle Stability.

The wide applications of Ni-rich LiNi1- x-y Cox Mny O2 cathodes are severely limited by capacity fading and voltage fading during the cycling process resulting from the pulverization of particles, interfacial side reactions, and phase transformation. The canonical surface modification approach can improve the stability to a certain extent; however, it fails to resolve the key bottlenecks. The preparation of Li(Ni0.4 Co0.2 Mn0.4 )1- x Tix O2 on the surface of LiNi0.8 Co0.1 Mn0.1 O2 particles with a coprecipitation method is reported. After sintering, Ti diffuses into the interior and mainly distributes along surface and grain boundaries. A strong surface and grain boundary strengthening are simultaneously achieved. The pristine particles are fully pulverized into first particles due to mechanical instability and high strains, which results in serious capacity fading. In contrast, the strong surface and the grain boundary strengthening can maintain the structural integrity, and therefore significantly improve the cycle stability. A general and simple strategy for the design of high-performance Ni-rich LiNi1- x - y Cox Mny O2 cathode is provided and is applicable to surface modification and grain-boundary regulation of other advanced cathodes for batteries.

Comparison of protein expression profiles of the hepatopancreas in Fenneropenaeus chinensis challenged with heat-inactivated Vibrio anguillarum and white spot syndrome virus.

Fenneropenaeus chinensis (Chinese shrimp) culture industry, like other Penaeidae culture, has been seriously affected by the shrimp diseases caused by bacteria and virus. To better understand the mechanism of immune response of shrimp to different pathogens, proteome research approach was utilized in this study. Firstly, the soluble hepatopancreas protein samples in adult Chinese shrimp among control, heat-inactivated Vibrio-challenged and white spot syndrome virus-infected groups were separated by 2-DE (pH range, 4-7; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and pH range, 3-10; tricine-SDS-PAGE). Then the differentially expressed protein spots (≥1.5-fold or ≤0.67-fold averagely of controls) were analyzed by LC-ESI-MS/MS. Using Mascot online database searching algorithm and SEQUEST searching program, 48 and 49 differentially expressed protein spots were successfully identified in response to Vibrio and white spot syndrome virus infection, respectively. Based on these results, we discussed the mechanism of immune response of the shrimp and shed light on the differences between immune response of shrimp toward Vibrio and white spot syndrome virus. This study also set a basis for further analyses of some key genes in immune response of Chinese shrimp.

Effects of seawater salinity and temperature on growth and pigment contents in Hypnea cervicornis J. Agardh (Gigartinales, Rhodophyta).

This study simulated outdoor environmental living conditions and observed the growth rates and changes of several photosynthetic pigments (Chl a, Car, PE, and PC) in Hypnea cervicornis J. Agardh (Gigartinales, Rhodophyta) by setting up different ranges of salinity (25, 30, 35, 40, 45, and 50) and temperature (15, 20, 25, and 30°C). At conditions of culture, the results are as follows. (1) Changes in salinity and temperature have significant effects on the growth of H. cervicornis. The growth rates first increase then decrease as the temperature increases, while growth tends to decline as salinity increases. The optimum salinity and temperature conditions for growth are 25 and 25°C, respectively. (2) Salinity and temperature have significant or extremely significant effects on photosynthetic pigments (Chl a, Car, PE, and PC) in H. cervicornis. The results of this study are advantageous to ensure propagation and economic development of this species in the southern sea area of China.

Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis.

Complementary DNA (cDNA) and genomic DNA, including flanking regions of the chitinase gene (Fcchi-3) of Fenneropenaeus chinensis, were cloned and sequenced. Fcchi-3 was found to have 92.0 and 91.4% identity at the cDNA level to that of Litopenaeus vannamei and Marsupenaeus japonicus, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of L. vannamei (96.8%) and M. japonicus (93.4%). Based on the cDNA sequence, the genomic structure of the gene was characterized. Sequence analysis revealed that the Fcchi-3 gene was composed of seven exons with 411, 252, 186, 132, 171, 117 and 135 bp and six introns with 232, 196, 121, 90, 159 and 157 bp. Analysis by RT-PCR revealed that Fcchi-3 was a hepatopancreas specific gene. Semi-quantitative RT-PCR analysis revealed that Fcchi-3 transcript was down-regulated significantly in response to the challenge of WSSV at 5 h post-injection and then came back to normal level at 37 h. A fusion protein containing Fcchi-3 was produced and the purified recombinant protein exhibited similar biological function. The result of identification through LC-ESI-MS showed that three peptide fragments (-MAADPVLR-, -ATIDPAYNVPELSK- and -AILAVGGWNEGSPK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei chitinase-3. The recombinant Fcchi-3 could degrade the colloid chitin confirming that the recombinant protein is actually the chitinase.

Comparative proteomic profiles of the hepatopancreas in Fenneropenaeus chinensis response to hypoxic stress.

Hypoxia, as one suboptimal environmental condition, can affect the physiological state of shrimp during pond aquaculture. To better understand the mechanism of response to hypoxic stress in Chinese shrimp Fenneropenaeus chinensis, proteome research approach was utilized. Differentially expressed proteins of hepatopancreas in adult Chinese shrimp between the control and hypoxia-stressed groups were screened. By 2-DE analysis, 67 spots showed obvious changes after hypoxia. Using LC-ESI-MS/MS, 51 spots representing 33 proteins were identified including preamylase, arginine kinase, phosphopyruvate hydratase, citrate synthase, ATP synthase alpha subunit, chymotrypsin BI, chitinase, ferritin, C-type lectin receptors, transketolase, formylglutathione hydrolase, formyltetrahydrofolate dehydrogenase, aldehyde dehydrogenase, glutathione peroxidase, cytosolic manganese superoxide dismutase, protein disulfide isomerase, beta-actin, oncoprotein nm23, crustacyanin-C1 and so on. These proteins could be functionally classified into several groups such as proteins related to energy production, metabolism-related proteins, immune-related proteins, antioxidant proteins, chaperones, cytoskeleton proteins and ungrouped proteins. The transcription levels of ten selected genes encode the identified proteins were analyzed by real-time PCR at different sampling times of hypoxia. This study is the first analysis of differentially expressed proteins in the hepatopancreas of shrimp after hypoxia and provides a new insight for further study in hypoxic stress response of shrimp at the protein level.

Cloning and characterization of a novel C-type lectin gene from shrimp Litopenaeus vannamei.

In invertebrates, C-type lectins play crucial roles in innate immunity responses by mediating the recognition of host cells to pathogens and clearing microinvaders, which interact with carbohydrates and function as pattern recognition receptors (PRRs). A novel C-type lectin gene (LvLec) cDNA was cloned from hemocytes of Litopenaeus vannamei by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of LvLec was of 618 bp, consisting of a 5'-terminal untranslated region (UTR) of 60 bp and a 3'-UTR of 87 bp with a poly (A) tail. The deduced amino acid sequence of LvLec possessed all conserved features critical for the fundamental structure, such as the four cysteine residues (Cys(53), Cys(128), Cys(144), Cys(152)) involved in the formation of disulfides bridges and the potential Ca(2+)/carbohydrate-binding sites. The high similarity and the close phylogenetic relationship of LvLec shared with C-type lectins from vertebrates and invertebrates. The structural features of LvLec indicated that it was an invertebrate counterpart of the C-type lectin family. The cDNA fragment encoding the mature peptide of LvLec was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvLec) could agglutinate bacteria E. coli JM109 depending on Ca(2+), and the agglutination could be inhibited by mannose and EDTA. These results indicated that LvLec was a new member of C-type lectin family and involved in the immune defence response to Gram negative bacteria in Litopenaeus vannamei.

Molecular cloning, expression of a peroxiredoxin gene in Chinese shrimp Fenneropenaeus chinensis and the antioxidant activity of its recombinant protein.

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions in intracellular signal transduction. A Prx gene was firstly isolated in the crustacean, Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA consists of 942bp with a 594bp open reading frame, encoding 198 amino acids. The molecular mass of the deduced amino acid is 22041.17Da with an estimated pI of 5.17. Sequence comparison showed that Prx of F. chinensis shares 76%, 73% and 72% identity with that of Aedes aegypti, Branchiostoma belcheri tsingtaunese and Drosophila melanogaster, respectively. Northern blot analysis revealed the presence of Prx transcripts of F. chinensis in all tissues examined. Real-time PCR analysis indicated that the Prx showed different expression profiles in shrimp hemocytes and hepatopancreas after artificial infection with Vibrio anguillarum. In addition, a fusion protein containing Prx was produced in vitro. LC-ESI-MS analysis showed that four peptide fragments of the recombinant protein were identical to the corresponding sequence of F. chinensis Prx. And the purified recombinant proteins were shown to reduce H(2)O(2) in the presence of dithiothreitol.