To be or not to be planktonic? Self‐inhibition of biofilm development

The transition between planktonic growth and biofilm formation represents a tightly regulated developmental shift that has substantial impact on cell fate. Here, we highlight different mechanisms through which bacteria limit their own biofilm development. The mechanisms involved in these self‐inhibition processes include: (i) regulation by secreted small molecules, which govern intricate signalling cascades that eventually decrease biofilm development, (ii) extracellular polysaccharides capable of modifying the physicochemical properties of the substratum and (iii) extracellular DNA that masks an adhesive structure. These mechanisms, which rely on substances produced by the bacterium and released into the extracellular milieu, suggest regulation at the communal level. In addition, we provide specific examples of environmental cues (e.g. blue light or glucose level) that trigger a cellular response reducing biofilm development. All together, we describe a diverse array of mechanisms underlying self‐inhibition of biofilm development in different bacteria and discuss possible advantages of these processes.     

Pregnancy and virologic response to antiretroviral therapy in South Africa

Background

Although women of reproductive age are the largest group of HIV-infected individuals in sub-Saharan Africa, little is known about the impact of pregnancy on response to highly active antiretroviral therapy (HAART) in that setting. We examined the effect of incident pregnancy after HAART initiation on virologic response to HAART.

Methods and Findings

We evaluated a prospective clinical cohort of adult women who initiated HAART in Johannesburg, South Africa between 1 April 2004 and 30 September 2009, and followed up until an event, death, transfer, drop-out, or administrative end of follow-up on 31 March 2010. Women over age 45 and women who were pregnant at HAART initiation were excluded from the study; final sample size for analysis was 5,494 women. Main exposure was incident pregnancy, experienced by 541 women; main outcome was virologic failure, defined as a failure to suppress virus to ≤400 copies/ml by six months or virologic rebound >400 copies/ml thereafter. We calculated adjusted hazard ratios using marginal structural Cox proportional hazards models and weighted lifetable analysis to calculate adjusted five-year risk differences. The weighted hazard ratio for the effect of pregnancy on time to virologic failure was 1.34 (95% confidence limit [CL] 1.02, 1.78). Sensitivity analyses generally confirmed these main results.

Conclusions

Incident pregnancy after HAART initiation was associated with modest increases in both relative and absolute risks of virologic failure, although uncontrolled confounding cannot be ruled out. Nonetheless, these results reinforce that family planning is an essential part of care for HIV-positive women in sub-Saharan Africa. More work is needed to confirm these findings and to explore specific etiologic pathways by which such effects may operate.     

SAXS and the working protein

In this issue of Structure, Davies et al., 2005, present shape reconstructions for the molecular motor p97 using small angle X-ray scattering (SAXS) and offer insights into how ATP consumption is coupled to cyclical domain motions. This work emphasizes the emerging potential of SAXS for visualizing the workings of biological machines in solution.     

Distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data

Background

Pterygium is a common ocular surface disease characterized by fibrovascular invasion of the cornea and is sight-threatening due to astigmatism, tear film disturbance, or occlusion of the visual axis. However, the mechanisms for formation and post-surgical recurrence of pterygium are not understood, and a valid animal model does not exist. Here, we investigated the possible mechanisms of pterygium pathogenesis and recurrence.

Methods

First we performed a genome wide expression analysis (human Affymetrix Genechip, >22000 genes) with principal component analysis and clustering techniques, and validated expression of key molecules with PCR. The controls for this study were the un-involved conjunctival tissue of the same eye obtained during the surgical resection of the lesions. Interesting molecules were further investigated with immunohistochemistry, Western blots, and comparison with tear proteins from pterygium patients.

Results

Principal component analysis in pterygium indicated a signature of matrix-related structural proteins, including fibronectin-1 (both splice-forms), collagen-1A2, keratin-12 and small proline rich protein-1. Immunofluorescence showed strong expression of keratin-6A in all layers, especially the superficial layers, of pterygium epithelium, but absent in the control, with up-regulation and nuclear accumulation of the cell adhesion molecule CD24 in the pterygium epithelium. Western blot shows increased protein expression of beta-microseminoprotein, a protein up-regulated in human cutaneous squamous cell carcinoma. Gene products of 22 up-regulated genes in pterygium have also been found by us in human tears using nano-electrospray-liquid chromatography/mass spectrometry after pterygium surgery. Recurrent disease was associated with up-regulation of sialophorin, a negative regulator of cell adhesion, and never in mitosis a-5, known to be involved in cell motility.

Conclusion

Aberrant wound healing is therefore a key process in this disease, and strategies in wound remodeling may be appropriate in halting pterygium or its recurrence. For patients demonstrating a profile of 'recurrence', it may be necessary to manage as a poorer prognostic case and perhaps, more adjunctive treatment after resection of the primary lesion.     

Aspects of early arthritis. Traditional DMARD therapy: is it sufficient?

There is increasing evidence for beneficial effects of early DMARD (disease-modifying antirheumatic drug) therapy over delayed treatment in patients who present with arthritis of recent onset. However, no universal consensus exists concerning the choice of initial drug or whether single drugs or combinations should be given as initial treatments. Recent studies have focused on the benefits of various strategies in which treatments were tailored to achieve low levels of disease activity, as assessed using validated response criteria. These studies demonstrated superiority of 'aggressive' over 'conventional' approaches. Whether the inclusion of tumour necrosis factor antagonists or other biologic targeted therapies in such strategies confers additional benefits in terms of improved long-term outcomes must be clarified by further studies. Assessment of risks in the individual patient, allowing individual 'tailoring' of the initial treatment, would be desirable.     

Diagnosis of triple negative breast cancer using expression data with several machine learning tools

Breast cancer is one of the top three commonly caused cancers worldwide. Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, lacks expression of the oestrogen receptor, progesterone receptor, and HER2. This makes the prognosis poor and early detection hard. Therefore, AI based neural models such as Binary Logistic Regression, Multi-Layer Perceptron and Radial Basis Functions were used for differential diagnosis of normal samples and TNBC samples collected from signal intensity data of microarray experiment. Genes that were significantly upregulated in TNBC were compared with healthy controls. The MLP model classified TNBC and normal cells with anaccuracy of 93.4%. However, RBF gave 74% accuracy and binary Logistic Regression model showed an accuracy of 90.0% in identifying TNBC cases.     

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88 μM BAP with 108.6 μM adenine sulphate and 11.84 μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84 μM IBA, with 81.1 % rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.     

Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses

The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses.Deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV) are single-stranded positive-sense RNA viruses of the order Picornavirales and are regularly detected in honeybee populations in the United Kingdom (1). ABPV has been assigned to the family Dicistroviridae and is known to follow a classic acute-type infection strategy since relatively low loads (10³ to 10⁶ viruses per honeybee) can rapidly translate into overt symptoms of paralysis and ultimately death for the honeybee, depending on the mode of transmission (6, 33). ABPV shares >92% sequence homology with other members of the family Dicistroviridae, Kashmir bee virus and Israeli acute paralysis virus, across the eight conserved domains of the RNA-dependent RNA polymerase gene, and it has been proposed that these viruses have recently diverged and are variants of each other (7). Advances in the study of this proposed ABPV complex is revealing the significant impact these viruses may have on honeybee colonies on a global scale. For example, a recent study in the United States has observed a correlation between Israeli acute paralysis virus and colony collapse disorder (17). That said, other agents, including bacteria and microsporidia, have also been proposed as important factors in the onset of colony loss (25, 27).BQCV is similar to ABPV in that it, too, follows a typical acute infection strategy. This virus is known to infect honeybee queen cell larvae, causing the larvae to discolor and die (5). It has been shown to be associated with the microsporidian Nosema apis (4) although whether N. apis has a direct role in the transmission of this virus still needs to be determined. Both ABPV and BQCV have been detected in worker honeybees and pupae (38), and the viruses are transmitted orally, via food and feeding activities (14). BQCV has also been detected in queen honeybees (13), suggesting that vertical transmission is also important for this virus. Both BQCV (12) and ABPV (38) have been detected within the varroa mite; however, only ABPV (9) has been shown to be vectored by varroa mites and has been found associated with dead colonies infested with varroa mites in Germany, Russia, and the United States (1). Later modeling work (33) indicated that very large (10,000+) mite populations are required to kill a colony since it is difficult for ABPV to become established among the bee population due to its high virulence.DWV is currently designated as a member of the unassigned genus Iflavirus within the order Picornavirales. It is generally considered as less virulent than ABPV or Kashmir bee virus, but it is known to cause overt symptoms of wing deformities in developing honeybees, resulting in emerging honeybees that are unable to fly and die shortly (5). It is also speculated that a cloud of DWV sequence variants exists that have evolved from a common ancestor. This is due to the high sequence similarities DWV isolates share with Kakugo virus and Varroa destructor virus within the RNA-dependent RNA polymerase gene, yet differences in virus epidemiology and pathological effects distinguish them from each other (29). DWV has been detected in worker honeybees, pupae, larvae, drones, and queens (15, 18, 20) as well as within the varroa mite (38, 43) and more recently the mite Tropilaelaps mercedesae (21), implying a range of horizontal and vertical transmission routes. Despite their global occurrence, it is generally accepted that DWVs play a secondary role in the causes of honeybee disease compared to their parasitic and bacterial counterparts as the viruses routinely reside at low levels in colonies, with symptomatic infections being rare (5). Moreover, multiple variants with differing infection strategies can account for a lack of discernible symptoms.Whether these viruses follow a persistent, latent, inapparent, or progressive infection strategy still remains unclear. Persistent (often called chronic) infections imply that the rate of infection within a host is in balance with the reproduction rate of the infected cell type or host itself. This is achieved through a combination of changing virus replication and host immune responses. Latent infections occur when the virus lies dormant within the host (replication inactive) until activation by defined stimuli. Progressive infections are caused by viruses that enter the host cell and replicate undetected for many cellular generations over many years before manifesting overt or acute symptoms. These three infection strategies all evade the host immune system, which results in the inability of the host to fully expel the virus, and this inability is often lethal. Inapparent (often referred to as covert) infections are indicative of a highly evolved relationship between the virus and natural host. Moreover, these infections are distinct in that the natural host can eventually clear itself from this short-term infection (19). Infections of DWV are often described as inapparent (15); however, Yue et al. (44) have suggested that a distinction should be made between “true inapparent” and their newly defined “covert infection” based on the long-term nature of DWV infection in honeybee colonies and on the nature of its transmission. This conclusion is congruent with current knowledge that traditional serological screening methods for DWV have limitations in their sensitivity (20). Therefore, the presence and duration of DWV within colonies have often been underestimated using serological assays as the overt symptoms of the deformed wing phenotype (>10¹¹ virions per honeybee) are short-lived. Advances in virus detection methodologies have enabled the development of more sensitive techniques, such as PCR, and this has demonstrated that DWV persists for longer periods within colonies (38). However, based on the current research evidence, a case could be made that DWV actually follows the classic persistent infection strategy.DWV is thought to have an intricate relationship with varroa mites such that immunosuppression of the honeybee pupae by the mites results in increased DWV amplification when the honeybees are exposed to other pathogens (42). It has additionally been shown that the number of mites parasitizing honeybee pupae is positively correlated with the probability of their developing malformed wings (10). Other findings indicate that DWV replication within the mite and subsequent transmission to developing honeybees lead to the increased likelihood of the bees'' emerging with wing deformities (24, 43). Taken together, the expectation is that DWV-associated colony collapse would typically occur in the presence of a large (>2,000) varroa mite infestation carrying high levels of DWV and with a high proportion of deformed honeybees. While the effect of varroa mite-induced DWV disease is well recognized, i.e., wing deformities coupled with downregulation of immunity-related genes and antimicrobial peptides (36, 42) and impaired learning behavior (28), the impact of non-varroa mite-vectored DWV within asymptomatic honeybees still needs to be realized. Moreover, it was recently reported that varroa mite-free bumblebees that tested positive for DWV actually showed symptoms of DWV infection (23). Even though these bumblebees were in close proximity to DWV-infected and varroa mite-infested honeybee colonies, it is evidence that the dependency of DWV on varroa mite vectoring for a symptomatic infection (manifested as classic wing deformities or other symptoms) may not be as critical as previously thought.The purpose of this study was to investigate asymptomatic viral dynamics within husbanded honeybee colonies over an annual cycle. We set out to observe the relationship, if any, between virus infections, varroa mite parasitism and vectoring, honeybee colony health, and colony longevity. For the first time, a quantitative analysis of three picorna-like honeybee viruses over the course of a year was undertaken for DWV, ABPV, and BQCV.     

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.