Histopathologic Observations of Eyes in Exenterated Orbits After Neoadjuvant Intra-Arterial Cytoreductive Chemotherapy for Adenoid Cystic Carcinoma of the Lacrimal Gland.

PURPOSE: To assess whether exenteration specimens obtained after neoadjuvant intra-arterial cytoreductive chemotherapy (IACC) for adenoid cystic carcinoma of the lacrimal gland demonstrate significant ocular histopathologic alterations that might preclude future pursuit of globe-preserving therapy. METHODS: Retrospective histopathologic analysis of globes in IACC-treated exenteration specimens among the same cohort of patients whose survival outcomes have been reported. RESULTS: Twenty patients had specimens available. Nineteen globes revealed no abnormalities of the iris, ciliary body, lens, retinal pigment epithelium, choroid, or chorioretinal vasculature. Eighteen globes showed no optic nerve abnormalities. One globe from a patient who refused exenteration until adenoid cystic carcinoma recurrence supervened demonstrated optic nerve edema with a peripapillary hemorrhage and cotton wool spot, as well as hemorrhage and necrosis within an extraocular muscle. Eighteen globes showed no retinal abnormalities attributable to intra-arterial chemotherapy. Three globes showed incidental retinal findings: 2 globes contained 1 to 2 small peripheral retinal hemorrhages and 1 had a pigmented retinal hole. Seven demonstrated mild, chronic extraocular muscle inflammation, and 13 had unremarkable musculature. The single patient who received IACC via the internal carotid rather than the external carotid artery developed ophthalmic artery occlusion with orbital apex syndrome prior to exenteration, and diffuse necrosis and hemorrhage were evident histopathologically. CONCLUSIONS: Neoadjuvant IACC does not cause significant histopathologic damage to key ocular structures or compromise visual function in patients receiving intra-arterial chemotherapy through the external carotid artery. However, delivering chemotherapy through the internal carotid artery may result in visually significant thrombotic vascular events. The generally benign histopathological findings in these exenteration specimens support the concept of IACC delivery through the external carotid system as the cornerstone of a future globe-preserving strategy for lacrimal gland adenoid cystic carcinoma.

Correction: Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis).

[This corrects the article DOI: 10.1371/journal.pone.0157237.].

Impact of Heat Stress on Cellular and Transcriptional Adaptation of Mammary Epithelial Cells in Riverine Buffalo (Bubalus Bubalis).

The present study aims to identify the heat responsive genes and biological pathways in heat stressed buffalo mammary epithelial cells (MECs). The primary mammary epithelial cells of riverine buffalo were exposed to thermal stress at 42°C for one hour. The cells were subsequently allowed to recover at 37°C and harvested at different time intervals (30 min to 48 h) along with control samples (un-stressed). In order to assess the impact of heat stress in buffalo MECs, several in-vitro cellular parameters (lactate dehydrogenase activity, cell proliferation assay, cellular viability, cell death and apoptosis) and transcriptional studies were conducted. The heat stress resulted in overall decrease in cell viability and cell proliferation of MECs while induction of cellular apoptosis and necrosis. The transcriptomic profile of heat stressed MECs was generated using Agilent 44 K bovine oligonucleotide array and at cutoff criteria of ≥3-or ≤3 fold change, a total of 153 genes were observed to be upregulated while 8 genes were down regulated across all time points post heat stress. The genes that were specifically up-regulated or down-regulated were identified as heat responsive genes. The upregulated genes in heat stressed MECs belonged to heat shock family viz., HSPA6, HSPB8, DNAJB2, HSPA1A. Along with HSPs, genes like BOLA, MRPL55, PFKFB3, PSMC2, ENDODD1, ARID5A, and SENP3 were also upregulated. Microarray data revealed that the heat responsive genes belonged to different functional classes viz., chaperons; immune responsive; cell proliferation and metabolism related. Gene ontology analysis revealed enrichment of several biological processes like; cellular process, metabolic process, response to stimulus, biological regulation, immune system processes and signaling. The transcriptome analysis data was further validated by RT-qPCR studies. Several HSP (HSP40, HSP60, HSP70, HSP90, and HSPB1), apoptotic (Bax and Bcl2), immune (IL6, TNFα and NF-kß) and oxidative stress (GPX1 and DUSP1) related genes showed differential expression profile at different time points post heat stress. The transcriptional data strongly indicated the induction of survival/apoptotic mechanism in heat stressed buffalo MECs. The overrepresented pathways across all time points were; electron transport chain, cytochrome P450, apoptosis, MAPK, FAS and stress induction of HSP regulation, delta Notch signaling, apoptosis modulation by HSP70, EGFR1 signaling, cytokines and inflammatory response, oxidative stress, TNF-alpha and NF- kB signaling pathway. The study thus identified several genes from different functional classes and biological pathways that could be termed as heat responsive in buffalo MEC. The responsiveness of buffalo MECs to heat stress in the present study clearly suggested its suitability as a model to understand the modulation of buffalo mammary gland expression signature in response to environmental heat load.

Matrix-based three-dimensional culture of buffalo mammary epithelial cells showed higher induction of genes related to milk protein and fatty acid metabolism.

Demanding transcriptomic studies in livestock animal species could be replaced by good in vitro models mimicking the function of mammary gland. Mammary epithelial cells (MEC) are the functional unit of the mammary gland. Extracellular matrix is known to be a key factor providing normal homeostasis in three-dimensional (3D) environment as important signals are lost when cells are cultured in two-dimensional (2D) environment. The aims of this study were to establish a buffalo mammary epithelial cells (BMECs) in 3D culture using extracellular matrix and to determine whether such a 3D culture model has different expression pattern than 2D counterpart. The purified MEC generated after several passages were used to establish 3D culture using Geltrex matrix. The expression of milk casein genes viz., alpha S1-casein (CSN1S1), alpha S2-casein (CSN1S2), beta-casein (CSN2), kappa-casein (CSN3); and fatty acid metabolism genes viz., butyrophilin (BTN1A1), glycerol-3-phosphate acyltransferase (GPAM), fatty acid-binding protein 3 (FABP3), and stearoyl-CoA desaturase (SCD) was assessed in 3D culture in comparison to traditional monolayer culture using qRT-PCR. Notable morphological differences were observed for BMECs grown in 3D culture in comparison to 2D culture. Morphologically, epithelial structures grown in Geltrex matrix (3D) environment showed enhanced functional differentiation in comparison to 2D culture. In 3D culture, lumen and dome-like structures were formed by day 5, whereas polarized acinus-like structure were formed within 15 days of culturing. The expression data showed higher mRNA induction of milk casein and fatty acid metabolism genes in 10-day-old 3D BMECs culture in comparison to 2D monolayer culture. The result suggests that 3D organization of epithelial cells has favorable effect on induction of milk and fatty acid metabolism-related genes. Therefore, matrix-based 3D culture of MEC that recapitulate the structural and functional context of normal tissues could provide a better in vitro model to understand the mammary gland functioning of buffaloes.

Peripheral blood mononuclear cells: a potential cellular system to understand differential heat shock response across native cattle (Bos indicus), exotic cattle (Bos taurus), and riverine buffaloes (Bubalus bubalis) of India.

Circulating leukocytes can be used as an effective model to understand the heat stress response of different cattle types and buffaloes. This investigation aimed to determine the temporal profile of HSPs (HSP40, HSP60, HSP70, and HSP90) expression in circulating peripheral blood mononuclear cells (PBMCs) of Murrah buffaloes, Holstein-Friesian (HF), and Sahiwal cows in response to sublethal heat shock at 42 °C. The viability data indicated HF PBMCs to be the most affected to the heat shock, whereas Sahiwal PBMCs were least affected, indicating its better survivability during the heat stress condition. The qRT-PCR expression data showed significant increase in mRNA expression of the analyzed HSPs genes after heat stimuli to the PBMCs under in vitro condition. In each case, the HSPs were most upregulated at 2 h after the heat stress. Among the HSPs, HSP70 was relatively more expressed followed by HSP60 indicating the action of molecular chaperones to stabilize the native conformation of proteins. However, PBMCs from different cattle types and buffaloes showed difference in the extent of transcriptional response. The level of expression of HSPs throughout the time period of heat stress was highest in buffaloes, followed by HF and Sahiwal cows. The higher abundance of HSP70 mRNA at each time point after heat stress showed prolonged effect of heat stress in HF PBMCs. The data presented here provided initial evidence of transcriptional differences in PBMCs of different cattle types and buffaloes and warrant further research.

Selection of stable reference genes in heat stressed peripheral blood mononuclear cells of tropically adapted Indian cattle and buffaloes.

Eleven reference genes from different functional categories were assessed for their stable expression pattern in heat stressed PBMCs across Indian buffalo and cattle using GeNorm, NormFinder and BestKeeper algorithms. As the first report, we suggest B2M, RPS9 and RPS15a as suitable reference genes for accurate normalization of PBMC transcript data while the ACTB gene is not recommended.

Identification of Appropriate Reference Genes for qRT-PCR Analysis of Heat-Stressed Mammary Epithelial Cells in Riverine Buffaloes (Bubalus bubalis).

Gene expression studies require appropriate normalization methods for proper evaluation of reference genes. To date, not many studies have been reported on the identification of suitable reference genes in buffaloes. The present study was undertaken to determine the panel of suitable reference genes in heat-stressed buffalo mammary epithelial cells (MECs). Briefly, MEC culture from buffalo mammary gland was exposed to 42 °C for one hour and subsequently allowed to recover at 37 °C for different time intervals (from 30 m to 48 h). Three different algorithms, geNorm, NormFinder, and BestKeeper softwares, were used to evaluate the stability of 16 potential reference genes from different functional classes. Our data identified RPL4, EEF1A1, and RPS23 genes to be the most appropriate reference genes that could be utilized for normalization of qPCR data in heat-stressed buffalo MECs.

The role of N dopant in inducing ferromagnetism in (ZnO)n clusters (n = 1-16).

The effect of nitrogen doping on the magnetic properties of (ZnO)(n) clusters (n = 1-16) has been investigated using spin polarized density functional theory. The total energy calculations suggest that N is more stable at the O site than at the Zn site in (ZnO)(n) clusters and induces a magnetic moment of 1 µ(B)/N atom. The N-Zn-N configuration is more stable than isolated N for 3D structures. The N dopants do not show any tendency for clustering. The binding energy is found to decrease with the increase in the number of N dopants. The magnetic moment increases gradually with the increase in the number of atoms with 1 µ(B)/N atom for n ≤ 4 and less than 1 µ(B)/N for n > 4. The local magnetic moment is mainly localized at the N site with a small magnetic moment induced at the O site. The presence of a Zn vacancy (V(Zn)) induced an additional magnetic moment of 2 µ(B) on the nearest O atoms. The N dopant prefers to form a N-V(Zn) pair. The combination of N and V(Zn) in 3D structures leads to a total magnetic moment of 3 µB. The Mulliken charge transfers from Zn to N and O in all N doped (ZnO)(n) clusters. The calculated results are consistent with existing experimental and theoretical results.

Transition metal induced magnetism in smaller fullerenes (Cn for n≤36).

The magnetic properties of 3d transition metals (TM) encapsulated inside smaller fullerenes ranging from C20 to C36 have been investigated using spin polarized density functional theory. The TM impurities stabilize asymmetrically at an off-center position for n≥28. The total magnetic moment (MM) of TM@Cn complexes are largely contributed by TMs and a small amount of MM of 0.12-0.50 µB is induced on the cage carbon atoms. The 3d TM atoms interact with C atoms of C20 and C28 cage ferromagnetically (FM) except for Ni@C28 which shows antiferromagnetic (AFM) interaction. The magnetic interactions change from FM to AFM in C32 cage for Ti, V, Cr and Mn. The MM gets quenched in Ni@Cn for n≥32. The total MM of Mn@Cn does not show any change although the nature of magnetic interactions changes from FM to AFM at n=32. Ti and V are the only TMs which show positive cohesive energy in all fullerenes considered. The smallest fullerene which can encapsulate all 3d TM are Cn for n≥32, consistent with available experimental and theoretical results.