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Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

Melanin is known to provide strong third-harmonic generation (THG) contrast in human skin. With a high concentration in basal cell cytoplasm, THG contrast provided by melanin overshadows other THG sources in human skin studies. For better understanding of the THG signals in keratinocytes without the influence of melanin, an in vivo THG microscopy (THGM) study was first conducted on vitiliginous skin. As a result, the THG-brightness ratio between the melanin-lacking cytoplasm of basal cells and collagen fibers is about 1.106 at the dermal–epidermal junctions of vitiliginous skin, indicating high sensitivity of THGM for the presence of melanin. We further applied the in vivo THGM to assist evaluating the therapeutic outcome from the histopathological point of view for those showed no improvement under narrowband ultraviolet B therapy based on the seven-point Physician Global Assessment score. Our clinical study indicates the high potential of THGM to assist the histopathological assessment of the therapeutic efficacy of vitiligo treatments.

While the nucleus-to-cytoplasmic (N:C) ratio has traditionally been used for assessing cell malignancy, most N:C measurement techniques are time-consuming and performed on thin histological sections, which prohibit assessment of three-dimensional cell structure. A combined ultrahigh frequency ultrasound (US) and photoacoustic (PA) technique was used to assess the size and N:C ratio of cultured cancer cells in three dimensions (3D). The diameters of the cells and their stained nuclei were obtained by fitting the power spectrum of backscattered US pulses and emitted PA waves, respectively, to well-established theoretical models. For comparison, an imaging flow cytometer (IFC) was also used to determine the two-dimensional cell and nucleus sizes from large cell populations using brightfield and fluorescence images, respectively. An N:C ratio was calculated for each cell using the quotient of the measured nucleus diameter and the total cell diameter. The mean N:C ratios calculated using the sound-based approach were 0.68, 0.66, and 0.54 for MCF-7, PC-3, and MDA-MB-231 cells, respectively, and were in good agreement with the corresponding values of 0.68, 0.67, and 0.68 obtained using the IFC. The combined US and PA technique, which assesses cellular N:C ratio in 3D, has potential applications in the detection of circulating tumor cells in liquid biopsies.

A membrane potential change in cells is accompanied with mechanical deformation. This electromechanical response can play a significant role in regulating action potential in neurons and in controlling voltage-gated ion channels. However, measuring this subtle deformation in mammalian cells has been a difficult task. We show a plasmonic imaging method to image mechanical deformation in single cells upon a change in the membrane potential. Using this method, we have studied the electromechanical response in mammalian cells and have observed the local deformation within the cells that are associated with cell–substrate interactions. By analyzing frequency dependence of the response, we have further examined the electromechanical deformation in terms of mechanical properties of cytoplasm and cytoskeleton. We demonstrate a plasmonic imaging approach to quantify the electromechanical responses of single mammalian cells and determine local variability related to cell–substrate interactions.

Background and Objective: Could a normal – but persistent – stress response to impeded axonal transport lead to late-onset Alzheimer’s disease (AD)? Our results offer an affirmative answer, suggesting a mechanism for the abnormal production of amyloid-β (Aβ), triggered by the slowed axonal transport at old age. We hypothesize that Aβ precursor protein (APP) is a sensor at the endoplasmic reticulum (ER) that detects, and signals to the nucleus, abnormalities in axonal transport. When persistently activated, due to chronically slowed-down transport, this signaling pathway leads to accumulation of Aβ within the ER. Methods and Results: We tested this hypothesis with the neuronal cell line CAD. We show that, normally, a fraction of APP is transported into neurites by recruiting kinesin-1 via the adaptor protein, Fe65. Under conditions that block kinesin-1-dependent transport, APP, Fe65 and kinesin-1 accumulate in the soma, and form a complex at the ER. This complex recruits active c-Jun N-terminal kinase (JNK), which phosphorylates APP at Thr668. This phosphorylation increases the cleavage of APP by the amyloidogenic pathway, which generates Aβ within the ER lumen, and releases Fe65 into the cytoplasm. Part of the released Fe65 translocates into the nucleus, likely to initiate a gene transcription response to arrested transport. Prolonged arrest of kinesin-1-dependent transport could thus lead to accumulation and oligomerization of Aβ in the ER. Conclusion: These results support a model where the APP:Fe65 complex is a sensor at the ER for detecting the increased level of kinesin-1 caused by halted transport, which signals to the nucleus, while concomitantly generating an oligomerization-prone pool of Aβ in the ER. Our hypothesis could thus explain a pathogenic mechanism in AD.

To develop an effective hybrid bioartificial liver (BAL) device, the material of the scaffold is very important to support hepatocytes that have both growth ability and hepatic differentiated functions. In this study we used paper (Kimwipe, Kimberly-Clark Corp., Roswell, GA, U.S.A.) as a scaffold. Primary hepatocytes isolated from a normal adult rat liver could proliferate on the paper. The secretion of albumin into culture medium by the cells on the paper increased with time in culture and, compared to the cells on dishes, the amount of 48 h albumin secretion at Day 10 was two times larger. Perpendicular sections of hepatocytes on the paper revealed that the cells fell into cavities made by intersecting fibers, piled up, and formed three to four layers. The piled-up cells changed their shape from flat to cuboidal and enlarged their cytoplasm, which was rich in organelles such as mitochondria and peroxisomes with a nucleoid. In addition, they formed bile canalicular structures between the cells. Their morphological appearance was similar to in vivo hepatocytes. Paper (Kimwipe) may be a good candidate as a scaffold to make a BAL device.

Considerable controversy exists concerning the origin and composition of Alzheimer neurofibrillary tangles (ANT) and of paired helical filaments (PHF), the abnormal cytoplasmic fibers which ultrastructurally are the major components of ANT. Thus far, the unusual solubility properties of PHF have hindered the analysis of ANT. A new procedure is presented for isolating purified PHF which are soluble in the presence of sodium dodecyl sulfate. The purification protocol involves differential and rate zonal centrifugation, treatment with the detergents sarcosyl and sulfobetain 3–14, and sonication. The isolated PHF from Alzheimer disease/senile dementia of the Alzheimer type (7 cases) and Down's syndrome (one case) have been characterized structurally by negative-stain electron microscopy, biochemically by PAGE, and immunologically by both the ELISA technique and Western blot analysis using a monoclonal antibody prepared against ANT. Distinct polypeptides were shown to be associated with this structure and not seen in preparations from young and age-matched normal brains.

The reactivity of rabbit antisera to rat lung secretory proteins with other rodent species was evaluated by immunocytochemistry. Rabbit anti-rat surfactant apoprotein antiserum reacts with the cytoplasm of rat, mouse, and hamster type II pneumocytes and is specific for these cells. Rabbit antiserum to rat Clara cell secretory proteins stains rat, mouse, and hamster Clara cells. Rabbit antisera specific to the two antigenic types of rat Clara cell antigens were also both reactive with rat, mouse, and hamster Clara cells. An antiserum to the non-serum proteins of hamster lung lavage was also prepared and shown to be specifically reactive with hamster Clara cells. The availability of specific reagents for secretory proteins of rodent lungs is expected to facilitate studies of the respective cell types in various pathologic states.

Antigenic competition is known to be a widespread phenomenon when using crude extracts of antigens (e.g., Escherichia coli cytoplasmic proteins) for immunization. This non-specific form of immune suppression can be partially overcome by passive immunization with antibodies against dominant antigens (which are the suppressive molecules) before injection of the antigenic mixture. Blocking these immunodominant antigens or antigenic determinants by a passively administered antibody permits antibody responses against hitherto weakly or non-immunogenic molecules.

Glycerol kinase and glycerophosphate dehydrogenase activities have been demonstrated in the obligate halophile Halobacterium cutirubrum. Both enzymes are stereospecific for the formation of only the sn-3-glycerophosphate, and are associated with the cell envelope and cytoplasmic fractions. The stereospecificity of these enzymes in the obligate halophile is thus the same as in nonhalophiles and in higher organisms. The bearing of these findings on the biosynthetic pathway for the di-O-phytanyl glycerol ether phospholipids in H. cutirubrum is discussed.

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembraneous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembraneous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The sub-membraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells. Cytoskeletal components are probably involved in the formation of cell contacts and regulation of contact areas between blastomeres, as well as the control of cell movement. Thus, nonerythroid spectrin and actin may play important roles in the regulation of cleavage-stage development and blastocyst transformation.