A Flexible Mouse-On-Mouse Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents,Immunofluorescence, and Multiple Antibody Staining

Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.     

STRETCHING OF THE SCIATIC NERVE—A Means of Relieving Postoperative Pain Following Removal of Ruptured Lumbar Intervertebral Discs

Stretching the sciatic nerve for the relief of “sciatica” was frequently employed before 1900 and was subsequently abandoned, probably because it was done without sufficient scrutiny of the indications. The procedure has recently been employed in cases in which “sciatica” remains following the operative removal of ruptured intervertebral discs, and it has been instrumental in relieving postoperative “sciatica” when the cause was the formation of adhesions about the lumbar nerve roots. If the nerve root is compressed by recurrent disc protrusion or by adjacent bone, the manipulation usually increases the pain, a phenomenon that has been helpful from a diagnostic standpoint.     

Roles of leader and follower cells in collective cell migration

Collective cell migration is a widely observed phenomenon during animal development, tissue repair, and cancer metastasis. Considering its broad involvement in biological processes, it is essential to understand the basics behind the collective movement. Based on the topology of migrating populations, tissue-scale kinetics, called the “leader–follower” model, has been proposed for persistent directional collective movement. Extensive in vivo and in vitro studies reveal the characteristics of leader cells, as well as the special mechanisms leader cells employ for maintaining their positions in collective migration. However, follower cells have attracted increasing attention recently due to their important contributions to collective movement. In this Perspective, the current understanding of the molecular mechanisms behind the “leader–follower” model is reviewed with a special focus on the force transmission and diverse roles of leaders and followers during collective cell movement.     

On the Temporal Characteristics of Performance Variability in Attention Deficit Hyperactivity Disorder (ADHD)

Increased intra-subject variability of reaction times (ISV-RT) is one of the most consistent findings in attention-deficit/hyperactivity disorder (ADHD). Although the nature of this phenomenon is still unclear, it has been hypothesised to reflect interference from the Default Mode Network (DMN). So far, ISV-RT has been operationally defined either as a frequency spectrum of the underlying RT time series, or as a measure of dispersion of the RT scores distribution. Here, we use a novel RT analysis framework to link these hitherto unconnected facets of ISV-RT by determining the sensitivity of different measures of RT dispersion to the frequency content of the underlying RT time series. N=27 patients with ADHD and N=26 healthy controls performed several visual N-back tasks. Different measures of RT dispersion were repeatedly modelled after individual frequency bands of the underlying RT time series had been either extracted or suppressed using frequency-domain filtering. We found that the intra-subject standard deviation of RT preserves the “1/f noise” characteristic typical of human RT data. Furthermore and most importantly, we found that the ex-Gaussian parameter τ is rather exclusively sensitive to frequencies below 0.025 Hz in the underlying RT time series and that the particularly slow RTs, which nourish τ, occur regularly as part of an quasi-periodic, ultra-slow RT fluctuation. Overall, our results are compatible with the idea that ISV-RT is modulated by an endogenous, slowly fluctuating process that may reflect DMN interference.     

Heat Generation/Absorption Effects in a Boundary Layer Stretched Flow of Maxwell Nanofluid: Analytic and Numeric Solutions

Analysis has been done to investigate the heat generation/absorption effects in a steady flow of non-Newtonian nanofluid over a surface which is stretching linearly in its own plane. An upper convected Maxwell model (UCM) has been utilized as the non-Newtonian fluid model in view of the fact that it can predict relaxation time phenomenon which the Newtonian model cannot. Behavior of the relaxations phenomenon has been presented in terms of Deborah number. Transport phenomenon with convective cooling process has been analyzed. Brownian motion “D_b” and thermophoresis effects “D_t” occur in the transport equations. The momentum, energy and nanoparticle concentration profiles are examined with respect to the involved rheological parameters namely the Deborah number, source/sink parameter, the Brownian motion parameters, thermophoresis parameter and Biot number. Both numerical and analytic solutions are presented and found in nice agreement. Comparison with the published data is also made to ensure the validity. Stream lines for Maxwell and Newtonian fluid models are presented in the analysis.     

Crossover Patterning by the Beam-Film Model: Analysis and Implications

Crossing-over is a central feature of meiosis. Meiotic crossover (CO) sites are spatially patterned along chromosomes. CO-designation at one position disfavors subsequent CO-designation(s) nearby, as described by the classical phenomenon of CO interference. If multiple designations occur, COs tend to be evenly spaced. We have previously proposed a mechanical model by which CO patterning could occur. The central feature of a mechanical mechanism is that communication along the chromosomes, as required for CO interference, can occur by redistribution of mechanical stress. Here we further explore the nature of the beam-film model, its ability to quantitatively explain CO patterns in detail in several organisms, and its implications for three important patterning-related phenomena: CO homeostasis, the fact that the level of zero-CO bivalents can be low (the “obligatory CO”), and the occurrence of non-interfering COs. Relationships to other models are discussed.     

Fueling the Flames: Mammalian Programmed Necrosis in Inflammatory Diseases

Programmed necrosis or necroptosis is an inflammatory form of cell death driven by TNF-like death cytokines, toll-like receptors, and antigen receptors. Unlike necrosis induced by physical trauma, a dedicated pathway is involved in programmed necrosis. In particular, a kinase complex composed of the receptor interacting protein kinase 1 (RIPK1) and RIPK3 is a central step in necrotic cell death. Assembly and activation of this RIPK1–RIPK3 “necrosome” is critically controlled by protein ubiquitination, phosphorylation, and caspase-mediated cleavage events. The molecular signals cumulate in formation of intracellular vacuoles, organelle swelling, internal membrane leakage, and eventually plasma membrane rupture. These morphological changes can result in spillage of intracellular adjuvants to promote inflammation and further exacerbate tissue injury. Because of the inflammatory nature of necrosis, it is an attractive pathway for therapeutic intervention in acute inflammatory diseases.Necrosis and tissue inflammation are two tightly linked phenomena. Cell injury induced by excessive trauma such as heat shock and osmotic shock can result in cell death with “necrotic morphology.” These relatively nonspecific means to trigger necrosis have contributed to the notion that necrosis is caused by excessive insults and does not involve elaborate intracellular signaling pathways. In contrast to the notion that necrosis is associated with harmful pathologies, recent work indicates that necrosis can have beneficial roles in certain biological responses. Proteomic approaches and RNA interference screens have identified several crucial regulators of necrosis induced by TNF-like death cytokines. Because a dedicated molecular circuitry is involved, the terms “programmed necrosis” and “necroptosis” have been used to distinguish these types of necrotic cell death from necrosis induced by physical trauma or insults. Here I will discuss the molecular pathway that regulates programmed necrosis/necroptosis. For the sake of simplicity, we will use the term necrosis to refer to programmed necrosis induced by defined death cytokines.     

Lamellar Thickness and Stretching Temperature Dependency of Cavitation in Semicrystalline Polymers

Polybutene-1 (PB-1), a typical semicrystalline polymer, in its stable form I shows a peculiar temperature dependent strain-whitening behavior when being stretched at temperatures in between room temperature and melting temperature of the crystallites where the extent of strain-whitening weakens with the increasing of stretching temperature reaching a minima value followed by an increase at higher stretching temperatures. Correspondingly, a stronger strain-hardening phenomenon was observed at higher temperatures. The strain-whitening phenomenon in semicrystalline polymers has its origin of cavitation process during stretching. In this work, the effect of crystalline lamellar thickness and stretching temperature on the cavitation process in PB-1 has been investigated by means of combined synchrotron ultrasmall-angle and wide-angle X-ray scattering techniques. Three modes of cavitation during the stretching process can be identified, namely “no cavitation” for the quenched sample with the thinnest lamellae where only shear yielding occurred, “cavitation with reorientation” for the samples stretched at lower temperatures and samples with thicker lamellae, and “cavitation without reorientation” for samples with thinner lamellae stretched at higher temperatures. The mode “cavitation with reorientation” occurs before yield point where the plate-like cavities start to be generated within the lamellar stacks with normal perpendicular to the stretching direction due to the blocky substructure of the crystalline lamellae and reorient gradually to the stretching direction after strain-hardening. The mode of “cavitation without reorientation” appears after yield point where ellipsoidal shaped cavities are generated in those lamellae stacks with normal parallel to the stretching direction followed by an improvement of their orientation at larger strains. X-ray diffraction results reveal a much improved crystalline orientation for samples with thinner lamellae stretched at higher temperatures. The observed behavior of microscopic structural evolution in PB-1 stretched at different temperatures explains above mentioned changes in macroscopic strain-whitening phenomenon with increasing in stretching temperature and stress-strain curves.     

The Molecular Signature of HIV-1-Associated Lipomatosis Reveals Differential Involvement of Brown and Beige/Brite Adipocyte Cell Lineages

Highly active antiretroviral therapy has remarkably improved quality of life of HIV-1-infected patients. However, this treatment has been associated with the so-called lipodystrophic syndrome, which conveys a number of adverse metabolic effects and morphological alterations. Among them, lipoatrophy of subcutaneous fat in certain anatomical areas and hypertrophy of visceral depots are the most common. Less frequently, lipomatous enlargements of subcutaneous fat at distinct anatomic areas occur. Lipomatous adipose tissue in the dorso-cervical area (“buffalo hump”) has been associated with a partial white-to-brown phenotype transition and with increased cell proliferation, but, to date, lipomatous enlargements arising in other parts of the body have not been characterized. In order to establish the main molecular events associated with the appearance of lipomatosis in HIV-1 patients, we analyzed biopsies of lipomatous tissue from “buffalo hump” and from other anatomical areas in patients, in comparison with healthy subcutaneous adipose tissue, using a marker gene expression approach. Both buffalo-hump and non-buffalo-hump lipomatous adipose tissues exhibited similar patterns of non-compromised adipogenesis, unaltered inflammation, non-fibrotic phenotype and proliferative activity. Shorter telomere length, prelamin A accumulation and SA-β-Gal induction, reminiscent of adipocyte senescence, were also common to both types of lipomatous tissues. Buffalo hump biopsies showed expression of marker genes of brown adipose tissue (e.g. UCP1) and, specifically, of “classical” brown adipocytes (e.g. ZIC1) but not of beige/brite adipocytes. No such brown fat-related gene expression occurred in lipomatous tissues at other anatomical sites. In conclusion, buffalo hump and other subcutaneous adipose tissue enlargements from HIV-1-infected patients share a similar lipomatous character. However, a distorted induction of white-to-“classical brown adipocyte” phenotype appears unique of dorso-cervical lipomatosis. Thus, the insults caused by HIV-1 viral infection and/or antiretroviral therapy leading to lipomatosis are acting in a location- and adipocyte lineage-dependent manner.     

Conscious Augmentation of Creative State Enhances “Real” Creativity in Open-Ended Analogical Reasoning

Humans have an impressive ability to augment their creative state (i.e., to consciously try and succeed at thinking more creatively). Though this “thinking cap” phenomenon is commonly experienced, the range of its potential has not been fully explored by creativity research, which has often focused instead on creativity as a trait. A key question concerns the extent to which conscious augmentation of state creativity can improve creative reasoning. Although artistic creativity is also of great interest, it is creative reasoning that frequently leads to innovative advances in science and industry. Here, we studied state creativity in analogical reasoning, a form of relational reasoning that spans the conceptual divide between intelligence and creativity and is a core mechanism for creative innovation. Participants performed a novel Analogy Finding Task paradigm in which they sought valid analogical connections in a matrix of word-pairs. An explicit creativity cue elicited formation of substantially more creative analogical connections (measured via latent semantic analysis). Critically, the increase in creative analogy formation was not due to a generally more liberal criterion for analogy formation (that is, it appeared to reflect “real” creativity rather than divergence at the expense of appropriateness). The use of an online sample provided evidence that state creativity augmentation can be successfully elicited by remote cuing in an online environment. Analysis of an intelligence measure provided preliminary indication that the influential “threshold hypothesis,” which has been proposed to characterize the relationship between intelligence and trait creativity, may be extensible to the new domain of state creativity.     

Drug Resistance in Natural Isolates of Leishmania donovani s.l. Promastigotes Is Dependent of Pgp170 Expression

Resistance of pathogens to drugs is a growing concern regarding many diseases. Parasites like Leishmania, Plasmodium and Entamoeba histolytica; and neoplastic cells, present the multidrug-resistant phenotype rendering chemotherapy ineffective. The acquired resistance of Leishmania to antimony has generated intense research on the mechanisms involved but the question has not yet been resolved. To test the hypothesis that drug efflux in Leishmania, as measured by flow cytometry using the fluorescent dye Rhodamine-123, is largely dependent on the number of efflux pumps an isolate can express, the amount of Pgp 170 molecules was assessed in ten field isolates (5 “resistant” and 5 “susceptible”) using: Western Blotting, Confocal and Transmission Electron Microscopy, and proteomics. Their survival after exposure to three antileishmanial drugs, in vitro, was evaluated and clinical data were compared to the in vitro results. All isolates were resistant to Glucantime but susceptible to Miltefosine, whilst Amphotericin B was more effective on the “susceptible” isolates. The MDR gene, expressing the transmembrane efflux pump Pgp 170, appears to play a key role in the phenomenon of drug resistance. When “susceptible” versus “resistant” parasites were compared, it was shown that the higher the number of Pgp 170 molecules the higher the Rhodamine-123 efflux from the parasite body and, when exposed to the drug, the number of efflux pumps increased. However, the rate of this increase was not linear and it is possible that there is a maximum number of Pgp 170 molecules an isolate can express. Nevertheless, the phenomenon is a complex one and other factors and proteins are involved in which the HSP-70 group proteins, detected in the “resistant” isolates, may play a significant role.     

Contrasting roles of dynamics in protein allostery: NMR and structural studies of CheY and the third PDZ domain from PSD-95

Allosteric regulation is a ubiquitous phenomenon exploited in biological processes to control cells in a myriad of ways. It is also of emerging interest in the design of functional proteins and therapeutics. Even though allostery was proposed over 50 years ago and has been studied intensively from a structural perspective, many key details of allosteric mechanisms remain mysterious. Over the last decade significant attention has been paid to the “dynamic component” of allostery, as opposed to the analysis of rigid structures. Nuclear magnetic resonance spectroscopy and its ability to detect conformationally dynamic processes at atomic resolution have played an important role in expanding our understanding of allosteric mechanisms and opening up new questions. This article focuses on work that highlights how protein dynamics can factor into allosteric processes in distinct ways. Two cases are contrasted. The first considers the “traditionally allosteric” protein CheY, which undergoes a conformational change as a key element of its allostery. The second considers the more rarely observed “dynamic allostery” in a PDZ domain, in which allosteric behavior arises from changes in internal structural dynamics. Interestingly, the dynamic processes in these two contrasting examples occur on different timescales. In the case of the PDZ domain, subsequent experimental and computational work is reviewed to reveal a more complete picture of this interesting case of allostery.     

Distractor Interference during a Choice Limb Reaching Task

According to action-centered models of attention, the patterns of distractor interference that emerge in selective reaching tasks are related to the time and effort required to resolve a race for activation between competing target and non-target response producing processes. Previous studies have only used unimanual aiming tasks and, as such, only examined the effects of competition that occurs within a limb. The results of studies using unimanual aiming movements often reveal an “ipsilateral effect” - distractors on the same side of space as the effector cause greater interference than distractors on the opposite side of space. The cost of the competition when response selection is between the limbs has yet to be addressed. Participants in the present study executed reaching movements to 1 of 4 (2 left, 2 right) possible target locations with and without a distractor. Participants made ipsilateral reaches (left hand to left targets, right hand to right targets). In contrast to studies using unimanual aiming movements, a “contralateral effect” was observed; distractors affording responses for the other hand (in contralateral space) caused more interference than distractors affording responses for the same hand. The findings from the present research demonstrate that when certain portions of response planning must be resolved prior to response initiation, distractors that code for that dimension cause the greatest interference.     

Immune Responses Elicited in Tertiary Lymphoid Tissues Display Distinctive Features

During chronic inflammation, immune effectors progressively organize themselves into a functional tertiary lymphoid tissue (TLT) within the targeted organ. TLT has been observed in a wide range of chronic inflammatory conditions but its pathophysiological significance remains unknown. We used the rat aortic interposition model in which a TLT has been evidenced in the adventitia of chronically rejected allografts one month after transplantation. The immune responses elicited in adventitial TLT and those taking place in spleen and draining lymph nodes (LN) were compared in terms of antibody production, T cell activation and repertoire perturbations. The anti-MHC humoral response was more intense and more diverse in TLT. This difference was associated with an increased percentage of activated CD4+ T cells and a symmetric reduction of regulatory T cell subsets. Moreover, TCR repertoire perturbations in TLT were not only increased and different from the common pattern observed in spleen and LN but also “stochastic,” since each recipient displayed a specific pattern. We propose that the abnormal activation of CD4+ T cells promotes the development of an exaggerated pathogenic immune humoral response in TLT. Preliminary findings suggest that this phenomenon i) is due to a defective immune regulation in this non-professional inflammatory-induced lymphoid tissue, and ii) also occurs in human chronically rejected grafts.     

Existence of abnormal protein aggregates in healthy Escherichia coli cells

Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. In addition, several groups have reported a virtual absence of protein aggregates in healthy cells. In contrast to previous studies and the expected outcome, we observed aggregated proteins in aerobic exponentially growing and “healthy” Escherichia coli cells. We observed overrepresentation of “aberrant proteins,” as well as substrates of the major conserved chaperone DnaK (Hsp70) and the protease ClpXP (a serine protease), in the aggregates. In addition, the protein aggregates appeared to interact with chaperones known to be involved in the aggregate repair pathway, including ClpB, GroEL, GroES, and DnaK. Finally, we showed that the levels of reactive oxygen species and unfolded or misfolded proteins determine the levels of protein aggregates. Our results led us to speculate that protein aggregates may function as a temporary “trash organelle” for cellular detoxification.     

Jumping Across the Gap - A Series of Atrial Extrastimuli

The “gap phenomenon” is an interesting phenomenon in electrophysiology arising from the differences in refractory periods at two or more levels of the atrioventricular (AV) conduction system. We present a patient with dual AV nodal physiology in whom the AH jump mediates the gap phenomenon. We also briefly discuss the other mechanisms of gap phenomenon that have been described in this setting.Key words: gap phenomenon, Atrial Extrastimuli     

SCINTIGRAMS OF THE THYROID GLAND—The Diagnosis of Morphologic Abnormalities with I131

Functioning thyroid tissue containing sufficient radioiodine can be visualized by scanning the gland with a directional scintillation counter.⁴ This visual representation of the gland is called a “scintigram.” Scintigrams have been invaluable in the detection and study of both “toxic” and non-functioning nodules, diffuse enlargement in hyperthyroidism and the subsequent reduction in gland size after treatment, carcinoma, and aberrant thyroid tissue.     

Mirrored Prominent Deck B Phenomenon: Frequent Small Losses Override Infrequent Large Gains in the Inverted Iowa Gambling Task

Since Bechara et al. pioneered its development, the Iowa Gambling Task (IGT) has been widely applied to elucidate decision behavior and medial prefrontal function. Although most decision makers can hunch the final benefits of IGT, ventromedial prefrontal lesions generate a myopic choice pattern. Additionally, the Iowa group developed a revised IGT (inverted IGT, iIGT) to confirm the IGT validity. Each iIGT trial was generated from the trial of IGT by multiplying by a “−” to create an inverted monetary value. Thus, bad decks A and B in the IGT become good decks iA and iB in the iIGT; additionally, good decks C and D in the IGT become bad decks iC and iD in the iIGT. Furthermore, IGT possessed mostly the gain trials, and iIGT possessed mainly the loss trials. Therefore, IGT is a frequent-gain–based task, and iIGT is a frequent-loss–based task. However, a growing number of IGT-related studies have identified confounding factors in IGT (i.e., gain-loss frequency), which are demonstrated by the prominent deck B phenomenon (PDB phenomenon). Nevertheless, the mirrored PDB phenomenon and guiding power of gain-loss frequency in iIGT have seldom been reexamined. This experimental finding supports the prediction based on gain-loss frequency. This study identifies the mirrored PDB phenomenon. Frequent small losses override occasional large gains in deck iB of the iIGT. Learning curve analysis generally supports the phenomenon based on gain-loss frequency rather than final outcome. In terms of iIGT and simple versions of iIGT, results of this study demonstrate that high-frequency loss, rather than a satisfactory final outcome, dominates the preference of normal decision makers under uncertainty. Furthermore, normal subjects prefer “no immediate punishment” rather than “final reward” under uncertainty.     

DNA methyltransferase-3–dependent nonrandom template segregation in differentiating embryonic stem cells

Asymmetry of cell fate is one fundamental property of stem cells, in which one daughter cell self-renews, whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms, such as plants, fungi, and mammals, has already been shown. However, before this current work, asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs), and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing, differentiating human and mouse ESCs. Moreover, we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3), indicating a molecular mechanism that regulates this phenomenon. Furthermore, our data support the hypothesis that retention of chromatids with the “old” template DNA preserves the epigenetic memory of cell fate, whereas localization of “new” DNA strands and de novo DNA methyltransferase to the lineage-destined daughter cell facilitates epigenetic adaptation to a new cell fate.