Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte.

In Drosophila melanogaster, the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the oocyte posterior end, while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. Past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, neither the molecular identity nor the nature of the signal is known. Here, we provide evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. We show that Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. Furthermore, we provide evidence that PFC contact maintains Par-1 and oskar mRNA localization and microtubule cytoskeleton polarity in the oocyte. Our observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.

Polarity Events in the Drosophila melanogaster Oocyte.

Cell polarity is a pre-requirement for many fundamental processes in animal cells, such as asymmetric cell division, axon specification, morphogenesis and epithelial tissue formation. For all these different processes, polarization is established by the same set of proteins, called partitioning defective (Par) proteins. During development in Drosophila melanogaster, decision making on the cellular and organism level is achieved with temporally controlled cell polarization events. The initial polarization of Par proteins occurs as early as in the germline cyst, when one of the 16 cells becomes the oocyte. Another marked event occurs when the anterior-posterior axis of the future organism is defined by Par redistribution in the oocyte, requiring external signaling from somatic cells. Here, we review the current literature on cell polarity events that constitute the oogenesis from the stem cell to the mature egg.

LEVEL OF PAIN AND ANALGESICS TAKEN AFTER CESAREAN SECTION AND DIFFERENCES ACCORDING TO TYPE OF ANESTHESIA AND DEMOGRAPHIC FACTORS.

The aim of this study was to investigate the level of pain and analgesic consumption in puerperas after cesarean section according to the type of anesthesia administered. This was a prospective study conducted at the Department of Obstetrics and Gynecology, Mostar University Hospital, in the period from September 2015 to June 2016. The study included 111 puerperas. Experimental group included 54 puerperas operated on under spinal anesthesia, while comparative group included 57 puerperas operated on under general anesthesia. Primary endpoints of the study were pain score and dose number of analgesics used. Input parameters of the study were age, gestational age, education, and place of residence. To determine the level of pain, visual analog scale for pain was used. Results showed that puerperas operated on under spinal anesthesia had significantly lower pain sensation (p=0.031) and less need for analgesic consumption in the postoperative period as compared to those operated on under general anesthesia (p=0.024). Increased age was associated with lower pain sensation (p=0.014) and need for analgesics (p<0.05). Higher level of education was associated with greater need for analgesics (p=0.016). Living in urban area was associated with greater pain sensation (p=0.023) and less need for analgesics (p<0.17). Spinal anesthesia for cesarean section resulted in less pain and less need for analgesics in the postoperative period compared to general anesthesia.

Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces.

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here, we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promote chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

Before cells divide to create copies of themselves, they need to duplicate their genetic material. To help split their DNA evenly, they build a machine called the mitotic spindle. The mitotic spindle is made of fine, tube-like structures called microtubules, which catch the chromosomes containing the genetic information and line them up at the center of the spindle. Microtubules push and pull the chromosomes by elongating or shortening their tips. But it remains unclear how the microtubules know when the chromosomes have reached center point. One way to find out is to remove proteins that accumulate in the middle of the spindle during division, such as the protein PRC1, which helps to assemble a subset of microtubules called bridging fibers, and the proteins Kif4A and Kif18A, which work like molecular rulers, shortening long microtubules. Usually, scientists would delete one of these proteins to see what impact this has. However, these experiments take days, giving the cell enough time to adapt and thus making it difficult to study the role of each of the proteins. Here, Jagric, Risteski, Martincic et al. used light to manipulate proteins at the exact moment of chromosome alignment and to move PRC1 from the spindle to the cell membrane. Consequently, Kif4A and Kif18A were removed from the spindle center. This caused the bridging fibers, which overlap with the microtubules that connect to the chromosomes, to become thinner. Jagric et al. discovered that without the molecular ruler proteins, the bridging fibers were also too long. This increased the overlap between the microtubules in the center of the spindle, causing the chromosomes to migrate away from the center. This suggests that the alignment of chromosomes in the middle of the spindle depends on the bridging microtubules, which need to be of a certain length to effectively move and keep the chromosomes at the center. Thus, forces that move the chromosomes are generated both at the tips of the microtubules and along the wall of microtubules. These results might inspire other researchers to reassess the role of bridging fibers in cell division. The optogenetic technique described here could also help to determine the parts other proteins have to play. Ultimately, this might allow researchers to identify all the proteins needed to align the chromosomes.

Pivot-and-bond model explains microtubule bundle formation.

During mitosis, microtubules form a spindle, which is responsible for proper segregation of the genetic material. A common structural element in a mitotic spindle is a parallel bundle, consisting of two or more microtubules growing from the same origin and held together by cross-linking proteins. An interesting question is what are the physical principles underlying the formation and stability of such microtubule bundles. Here we show, by introducing the pivot-and-bond model, that random angular movement of microtubules around the spindle pole and forces exerted by cross-linking proteins can explain the formation of microtubule bundles as observed in our experiments. The model predicts that stable parallel bundles can form in the presence of either passive crosslinkers or plus-end directed motors, but not minus-end directed motors. In the cases where bundles form, the time needed for their formation depends mainly on the concentration of cross-linking proteins and the angular diffusion of the microtubule. In conclusion, the angular motion drives the alignment of microtubules, which in turn allows the cross-linking proteins to connect the microtubules into a stable bundle.

Primary Care Provider Counseling Practices about Adverse Drug Reactions and Interactions in Croatia.

BACKGROUND: Prescribing medications is one of the most common medical decisions that is made by primary care providers (PCPs). In the Republic of Croatia, PCPs hold a key position in prescribing and evaluating the medications that are provided for patients. Accordingly, providing advice for patients regarding the potential adverse drug reactions (ADRs) and drug-drug interactions (DDIs) is frequently the responsibility of the PCPs. The aim of the current study was to assess the knowledge, attitudes, and counseling practices of PCPs regarding drug interactions and adverse effects. METHODS: After enrolling 195 PCPs that were selected at random, a survey was conducted while using an anonymous questionnaire that was created based on previously published studies, adjusted in a way that includes the most commonly prescribed medications in Croatia. RESULTS: Of the 10 questions on knowledge about DDIs and ADRs, the median number of correct responses by PCPs was 5 (interquartile range 4 to 7). More than half of respondents (56%) agreed with the claim that knowledge of drug side effects facilitated their work in family medicine. Almost all of the respondents (92.8%) explained side effects and drug interactions to special groups of patients (pregnant women, elderly patients etc.). CONCLUSION: The results show a need for additional education in the field of drug prescribing. However, PCPs were aware of the importance of counseling practices about adverse drug reactions and interactions and counseling practices among special patients populations are satisfactory.

Optogenetic reversible knocksideways, laser ablation, and photoactivation on the mitotic spindle in human cells.

At the onset of mitosis, cells assemble the mitotic spindle, a dynamic micromachine made of microtubules and associated proteins. Although most of these proteins have been identified, it is still unknown how their collective behavior drives spindle formation and function. Over the last decade, RNA interference has been the main tool for revealing the role of spindle proteins. However, the effects of this method are evident only after a longer time period, leading to difficulties in the interpretation of phenotypes. Optogenetics is a novel technology that enables fast, reversible, and precise control of protein activity by utilization of light. In this chapter, we present an optogenetic knocksideways method for rapid and reversible translocation of proteins from the mitotic spindle to mitochondria using blue light. Furthermore, we discuss other optical approaches, such as laser ablation of microtubule bundles in the spindle and creation of reference marks on the bundles by photoactivation of photoactivatable GFP. Finally, we show how different optical perturbations can be combined in order to acquire deeper understanding of the mechanics of mitosis.

Microtubule Sliding within the Bridging Fiber Pushes Kinetochore Fibers Apart to Segregate Chromosomes.

During cell division, mitotic spindle microtubules segregate chromosomes by exerting forces on kinetochores. What forces drive chromosome segregation in anaphase remains a central question. The current model for anaphase in human cells includes shortening of kinetochore fibers and separation of spindle poles. Both processes require kinetochores to be linked with the poles. Here we show, by combining laser ablation, photoactivation, and theoretical modeling, that kinetochores can separate without any attachment to one spindle pole. This separation requires the bridging fiber, a microtubule bundle that connects sister kinetochore fibers. Bridging fiber microtubules in intact spindles slide apart with kinetochore fibers, indicating strong crosslinks between them. We conclude that sliding of microtubules within the bridging fibers drives pole separation and pushes kinetochore fibers poleward by the friction of passive crosslinks between these fibers. Thus, sliding within the bridging fiber works together with the shortening of kinetochore fibers to segregate chromosomes.

Overlap microtubules link sister k-fibres and balance the forces on bi-oriented kinetochores.

During metaphase, forces on kinetochores are exerted by k-fibres, bundles of microtubules that end at the kinetochore. Interestingly, non-kinetochore microtubules have been observed between sister kinetochores, but their function is unknown. Here we show by laser-cutting of a k-fibre in HeLa and PtK1 cells that a bundle of non-kinetochore microtubules, which we term 'bridging fibre', bridges sister k-fibres and balances the interkinetochore tension. We found PRC1 and EB3 in the bridging fibre, suggesting that it consists of antiparallel dynamic microtubules. By using a theoretical model that includes a bridging fibre, we show that the forces at the pole and at the kinetochore depend on the bridging fibre thickness. Moreover, our theory and experiments show larger relaxation of the interkinetochore distance for cuts closer to kinetochores. We conclude that the bridging fibre, by linking sister k-fibres, withstands the tension between sister kinetochores and enables the spindle to obtain a curved shape.