Questions related to Plasmodium Falciparum
I am using primers from literature to detect the k1 allelic family in samples of Plasmodium falciparum. The expected range of product is 120-280bp, however, I have bright amplification around 500bp for some samples.
I'm not sure why this is in some samples while others are working fine
I did in silico study (molecular docking) of anti-malarial compounds' candidates that target the falciparin-2 and plasmodium falciparum dihydrofolate reductase-thymidylate synthase proteins and also in vitro study that target the plasmodium falciparum strain 3D7.
Interestingly, the in silico data and in vitro data are not consistent. Why these phenomenon could be happen? Are there any justification to discuss this phenomenon?
Thanks in advance
I am currently taking care of Plasmodium falciparum in vitro culture and in order for me to keep them alive, I was wondering how and when should I feed them if you have any protocol to advice me. I have access to complete media and RPMI.
Anyone can help me with this trouble shooting of plasmodium falciparum in vitro culture? I've been tried to culture plasmodium for approximately 2 months but they didn't grow well. Everytime I check thin blood smears of my culture, they appear like this (see the picture I attached) . It seems they couldn't invade or infect the red blood cells and stay outside. Please give me some advice about what I have to do so they can grow well and the ruptured schizont can invade RBCs.
Can someone assist me with analysis of sequence variations among Plasmodium falciparum variant with regards to how these variations might affect HRP2 produced by the available variants.
I am new to PCR. We are working on PCR based amplification of targeted DNA sequence of Plasmodium Falciparum. For that, We are targeting the Ring stage of the parasite. I want to know on which basis should i decide the gene for primer desinging.
I need to collect blood samples for microscopy of P. falciparum. Which tube is best for this? Heparin or plain vacutainer tubes? Will RBC lysis affect detection of the parasite? Thank you.
I should isolate single Plasmodium falciparum oocysts from mosquito midguts. In order to do that, I have to stain the midguts first. My present protocol is the following
1. a drop of 1% mercurochrome in PBS on a single midgut and incubation for 5 minutes
2. wash 3 times with PBS for 10 minutes each
3. mount on a slide and put a coverslip to flatten the sample for microscopy observation
For some reason, the oocysts are not stained as they should be (looking at pictures from other publications) and it's not possible to distinguish them by the rest of the sample. Everything has the same pink color.
Does anybody have some good detailed protocol to share? Also, my midgut samples (already dissected) are preserved in 70% ethanol. Could this affect the staining? Should I rehydrate them first or it doesn't matter?
Transfection of malaria parasites such as Plasmodium falciparum has been a very difficult experiment. Transfection efficiency is very low even through electroporation method. I wonder whether Lipofectamine 3000 reagent would help me transfect my malaria culture more easily. Please help me figure out.
I recently did a whole genome sequence on Plasmodium falciparum (Illumina , NGS). I have the data but i have been stuck at obtaining the sequences for specific genes of interest. Any tools or pipelines i can use for this? CLC Benchwork didnt work as the sequences are too big!
I am trying to perform AFM using plasmodium falciparum infected rbcs Using XE7 Park system, non contact mode. However i have no clue on how to differentiate between infected and non infected rbc as the system is not equiped with a microscope
Can anyone explain the procedure of RNA isolation from plasmodium falciaparum using direct-zol miniprep kit (zymo) ? And I need the information about plasmodium falciparum specification/condition ( culture volume, %parasitemia, ) as a starting material to isolate. Thank you
I am researching alternative methods to grow large numbers of Plasmodium falciparum gametocytes (for mosquito infections) which are not the traditional tissue culture flasks, and so can be maintained with minimal human labour if possible. Is anyone aware of any of these methods?
Would like to know the Plasmodium spp. image database that contains the labeled 5 kinds of species (falciparum, vivax, malariae, ovale, knowlesi) in 4 forms (ring, trophozoite, schizont, gametocyte). The image databases having some of the species and forms are also ok! The image number should be large enough to train a neural network (~1000)
I am about start conducting a research that involves detecting Plasmodium falciparum in human blood samples in a country located in West Africa. How will I know the best mRDT to use for my study? Please feel free to recommend with references (publications).
Hi I'm a Bioinformatics PhD student at UoL. I would like to chat to, or network with anyone who is knowledgeable on the cellular/molecular biology of P.falciparum or related strains/organisms such as T.gondii.
I have some very strange data/results which from my Bioinformatics/Comp Bio background does not make much sense in biological terms.
On plasmodb.org proteins like Stevor and Maurer's cleft which are host-parasite interaction proteins which I presume exist on the parasite cell surface membrane appear with GO annotations for the apicoplast organelle (as contain signal peptide).
I do know that these host-parasite interaction proteins as they are trying to evade the host immune system have different amino acid residue sequence behaviours (due to their evolution), and that homologue searching programs/servers like HMMER can pick up on this.
From a functional biological perspective what would the purpose be of these proteins residing in the apicoplast organelle which is predominantly of biosynthesis and export function be?
According to a few websites including from wikipedia, The organelle is an adaptation that the apicomplexan applies in penetration of a host cell.
Thus how more specifically in cell/molecular biology terms, how is the apicoplast organelle involved in the parasite invasion? Simplified answers only please as my cell biology/molecular biological knowledge is only intermediate.
I am looking for a plasmid for episomal expression in Plasmodium falciparum. Currently I am using pHC3 plasmid but no luck yet, also this plasmid has no map so difficult to handle it. A plasmid that has CAM promoter, supports for a complementation study and also has a full map, would be the ideal one. I appreciate a lot for any suggestion or sharing such plasmid if you are currently using it.
Thank you so much !
I have to amplify HVR-1 of Plasmodium falciparum ...I diluted primary PCR products 5X and 10X for nested PCR... and I got results in both..which one is more acceptable or effective...
I am trying to set up an experiment with a tightly synchronized P. falciparum culture to detect changes in expression levels of genes that are present during a particular stage of the parasite life cycle. I have attempted sorbitol synchronization and Percoll synchronization (and a combination of these two methods) and so far I've had no luck. My culture is either not synchronized enough, or I've manipulated it too much and inadvertently killed it.
Does anyone have a good synchronization protocol that produces a highly synchronized culture without killing off the culture? Any help would be immensely appreciated!
We now know that apicoplast (Trans) membrane proteins can originate from a vesicle and a signal peptide pathway. Normally signal peptide regions are cleaved out of the protein for apicoplast globular proteins and thus I'm assuming apicoplast membrane residing proteins no longer contain these signal peptide regions either? But I read in one paper that proteins targeted to the apicoplast via vesicles can still have these signal peptide regions within them which is confusing.
As the proteins will join the apicoplast membrane via vesicular fusion, will they not have their signal peptide regions cleaved off? so are there still apicoplast membrane proteins which exist in the these membranes with a signal peptide region within their sequence (which also contains a hydrophobic part)?
It is proposed, in order to reduce the osmotic stress within red blood cells after malaria infection, that plasmodium falciparum recjects nucleic acids out of the red blood cell.
Does anybody know which pathways are used for that?
I am running restriction digests for dhps gene particularly codons 540, 437 and 436 with the enzymes Fok1,Ava2 and mspA1 respectively. I have realized that with each digestion, the samples get cut but the controls (T9-96, 3D7 and IEC513/86 respectively) remain uncut or disappear altogether. Kindly advice on what could be the problem. Thank you.
i've been trying to grow my plasmodium falciparum in vitro since december last year but it seems that the parasites didn't want to grow. is there any recommendations for me to troubleshoot this problem? i already increase the amount of albumax and i used fresh blood every time i need to add blood into the culture.
I am busy with humanized mice for the study of PK/PD of antimalaria drugs. The work is iinvolving, engraftment of mice with human red blood cells (hRBCS), thereafter induce blood stage infection with plasmodium falciparum parasitized RBCs at a specific concentration. Knowing the concentration is important to monitor (by flow/elecron microscope) if the parasites growth reproductively in their artificial host. My question is to know, how to accurately work out this concentration using trucount tubes?
My question today is about generating free malaria parasite. I would like to know if someone is able to give me a protocol to extract this parasite from RBC,
using saponine ?
using tween (80 or 20) ?
using sorbitol ?
with concentration of ?
and without ultra-centrifugation if it is possible
ps: I have already read MMR 2013 by D. Mattei (chapter 4) so how do you replace Plasmion ?
I'm hoping someone with experience working with P. falciparum in vitro can help me with this!
I'm starting in vitro work with P. falciparum, and focusing on culturing gametocytes. I'll be working with strain 3D7.
Through my reading I've found that 3D7 is prone to losing it's gametocyte-formation ability when kept in culture for too long. A lot of papers suggest keeping cultures at a low passage number and continually thawing new feeder stocks to circumvent the problem.
My question is, what is considered a 'low' passage number? None of the papers I've read has given an actual number.
Thank you in advance for any help!
I have fresh dried blood spots that i need to extract Plasmodium falciparum parasite DNA for analysis of Antimalarial drugs Resistance markers.I will sequence the DNA thus i required high quality DNA.How do i go about the very first step ?
Something about the topic:
sample: whole cell lysate of plasmodium falciparum, 5ug/line;
gel: 12% Tris-Hcl SDS gel;
run: 80v for stacking, 130v for running gel;
stain: silverquest stain for total protein stain.
I saw vertical streak of my protein at the bottom area: I could still see the bands, but they were somehow separated.
The rest of proteins were well positioned as continuous horizontal bands.
It is not likely that I overload the well since it is only 5ug/line.
It is not likely that my samples had somewhat precipitation since I centrifuged them and the streaks only showed up at the bottom.
I have tried to detect Plasmodium falciparum lactate dehydrogenase directly from whole malaria infected blood using electrochemical impedance spectroscopy without success. I made dilution of the blood with buffer, PBS, and noninfected blood all to no avail. I wish anyone on this platform could be of help with an idea to solve the problem. Thank you
Please can anyone advise sourcing native plasmodium falciparum lactate dehydrogenase (also Pv-LDH). Does someone already have native purified protein(s) available? We are interested in a collaborative project to prepare more protein and / or direct purchase (2 - 10 mg) and looking to action this quickly.
Definitive host allows the parasite to reproduce sexually. Plasmodium sexually reproduce in the mosquito, then why it's an intermediate host?
I am collecting synchronized plasmodium falciparum samples, every 4 hours for the 48 hours life cycle. I would like to stagger the culture so I collection 2 time points, for a total of 24 hours. Can someone please point in me the right direction. I have read the following paper, but I am still left scratching my head as to how I should proceed.
Thank you in advance!!
I am trying to resolve plasmodium falciparum protein on a 10% sds GEL. i am using 0.1% saponin to lyse rbcs, further to which i add loading dye and heat @95 C for 10 min. but the bands are not resolving properly.
I have been trying this for weeks but I do not observe transgenic parasites emerge after drug selection. My protocol in brief is as follows:
(1) Designed gRNAs as 5'-G-N19-NGG-3' to be inserted into a pUF1-Cas9 "suicide vector" which kills the essential gene. (NGG is not included in actual gRNA sequence)
(2) Synthetic Donor plasmid containing left and right Homology arms (with tag included) and PAM sequence shielded by mutation.
(3) Circular plasmids are electroporated into Red Blood Cells (310 V, 1575 Ohm Resistance, and 950 uF capacitance).
(4) DNA-loaded Red Cells are infected with Plasmodium falciparum 3D7 for 2-3 generations.
(5) Drug selection (1.5 uM DSM1) is applied which selects only for Cas9 expression.
Please suggest any flaw in the procedure or suggest any better protocol to do this.
Thanks in Advance.
Hello all !
Would any of you be able to provide cDNA library of Plasmodium falciparum 3D7 under controlled circumstances? Or, are you aware of anyone who works on this parasite, that I could contact?
Of course, i will afford the shipment of the material.
Looking forward to hearing from you.
I am carrying out experiments to test the antimalarial activity of a range of iron chelators in vitro.
After quantification of parasitemia by flow cytometry, there was a base level of parasitemia present even at the highest concentrations administered, which I think may be due to the presence of gametocytes (still present in smears post-treatment)
I was wondering if there's a relatively simple way to remove the gametocytes.
Thanks in advance for any help!
We want to sequence P. falciparum (23 Mb) on a MiSeq with Nextera XT library prep. I have not worked with falciparum before but literature (see link) says that its extremely AT-biased genome makes library prep difficult because the Illumina polymerase does not effectively amplify AT-rich regions which results in uneven coverage. They suggest adding TMAC to the PCR and using Kapa Hifi as polymerase. However the paper is from 2012 and they did not use the Nextera Kit.
So my question is, has anyone experience with sequencing falciparum and/or has experience with tweaking the Nextera Kit.
Thanks a lot for your help.
We are studying Malaria drugs resistance, and we found that the % of early treatment failure in P. falciparum infected patients treated with ACT higher among children below 5 years.
Is this normal or explanable ?
I plan on culturing our p. falciparum parasites soon, and need a large amount of DNA (>10ug). This is much larger than I normally need, and I am not sure of how much culture I should grow up for this amount. I plan on extracting with a phenol/chloroform method. Does anyone have a suggestion on how much pRBC I should use and at what parasitaemia to grow my parasites to obtain such large amounts (and hopefully in a relativity short time frame)?
Thank you for your thoughts!!!
We are currently doing Plasmodium falciparum cultures (NF54 strain) in a perspective to infect Anopheles gambiae by membrane feeding. The Culture evolves up to gametocyte (stage V), but we are struggling to get positive infection by membrane feeding.
We get nice gametocytes in (Stage V) but they seem no infectious for the mosquitoes.
Im using in situ cell death detection kit-fluorescein roche for apoptosis detection in erythrocytic stage of Plasmodium falciparum. I used different protocols, but the problem is with fixation process, where I tried 4% paraformaldehyde as it is recommended, but the cells destroyed immediately after treatment with 0.1% triton X-100!!!!! I also tried 70% ice cold ethanol, but the blood sample clotted !!!! then I used 4% formadehyde with 0.1% glutaraldehyde, it gave some interesting result, but still some green background which should not be observed !!!!
Why we use Albumax during the preparation of complete RPMI media for P. falciparum and what is the function of albumax ?
So far, I have only seen evidence in avian malaria, but melanization, not total clearance; see:
Hillyer, J.F. et al. (2003) Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti. J. Parasitol. 89, 62–69
I'm doing the in vitro culture of Plasmodium falciparum (iRBC) with Albumax-I, would like to estimate the phagocytic activity of THP-1 derived macrophages towards iRBC, the literature which I have found were opsonized the iRBC with homologus serum to estimate the phagocytic activity but I am using variable blood groups to grow the culture I couldnt found the homologous serum in this context do the opsonization require or can I directly incubate the pre stained iRBC along with macrophage to estimate the phagocytosis by FACs
I am trying to get my plasmodium falciparum parasites synchronized to a 1hr time frame. I do a treatment of 5% sorbitol at 15hpi, then repeat at 5hpi. At 48hpi I run them through a MACs column to purify bursting schizonts, allow to grow for 1 hour and purify with sorbitol to get young rings. It seems as though my sorbitol treatments are too efficient because I still have schizonts and trophs in my culture when I should only have the young rings. Any have suggestions on how to make my window of synchronization tighter without putting stress on the parasites? Thank you in advance!
We have the DNA sequence, and the DNA extract of Plasmodium falciparum from which the DNA was sequenced.
We need the pure protein from the sequences fro immunological work. We lack the technology to express the proteins. Can anyone give us help?
Im trying to measure lipid peroxidation using fluorescent probe BODIPY FL C16 (Molecular Probes) using flow cytometry in plasmodium falciparum infected erythrocytes. But Im having troubles analyzing my results. I found that both green and red fluorescence decrease after my induction treatments comparing to control, but my intensity ratios increase. I found in literature ambiguous ways to analyze same data. Some just analyze an increment in green/red ratio, others look for an increase in green fluorescence and others looks for a decrease in green fluorescence. Im a little confused. If anyone has some experience with this dye and any suggestions they will be well received.
Thanks in advance!
I have done whole transcriptome sequencing for Plasmodium falciparum (2 strains) and it is compared. Can anyone tell me how to analyze the data completely, like based on what and how to interpret the result? Thanks in advance.
I would like to co-culture Plasmodium falciparum (pf) with mice cell line in this case will infection occur or can I separate the pf antigen for infection to mice cell line
What is the gene of target and appropriate primers for dtection of drugs resistance in plasmodium falciparum?
We recently received some Plasmodium falciparum sporozoites cryovials which are frozen with HES (hydroxy ethyl starch),Trehalose and DOPC (Dioleoylphosphatidyl-Choline) based freezing solution. As we have no experience with these freezing solution, can I please ask if anyone can advice us what principle should we follow when we thaw these cells (also would be fantastic if anyone can provide us a thawing protocol for similar conditions)?
Hello, I am planning to carry out an experiment involving Plasmodium falciparum infected Anopheles mosquito. For this, I have to artificially infect the mosquito with human blood containing gametocytes, after collecting the blood in to heparinized tube. So, in what condition should i keep the sample to prevent exflagellation of the microgametes for successful infection of the mosquitoes and for how long can I keep it before carrying out the experimental infection?
I am trying to amplify long genes (6Kb-12Kb) using cDNA as template. The RT I used was the SuperScript III with cDNA synthesis capacity is up to 12Kb, the template is an AT-rich genome.
I did check the RNA quality by measuring the 260:30 and 260:28 ratio, and is ok from 1.8 to 2.0. I treated the RNA with DNaseI previous to the RT and I tested by qPCR to confirm the cDNA synthesis using a housekeeping gene.
I did use one HiFi TaKaRa Taq polymerase which theoretically amplifies long fragments. I optimized the PCR protocol on genomic DNA and I confirm the amplification of the large fragments of my interest, now when I try the same protocol used on genomic DNA I do not get amplification.
Does anybody have experiences in long rage PCR using cDNA as template? Can somebody help me with suggestions?
Thanks a lot, those experiments constituted the last ones to finish my thesis and I don’t want to be stucked in this rut.
I am carrying out a research project to investigate the dominant circulating variants of the pfAMA1 gene in a population. I have already extracted the plasmodium falciparum gene but need to carry out PCR and sequencing to identify frequency of the different variants. I however need to know the region which exhibits the 3 variants and the length in base pairs so as to perform nested PCR and also determine the cost of sequencing according to the number of base pairs thus preventing wastage
I am trying to estimate the risk of P falciparum at the community level. I am aware of what has been done globally and regionally. Are there any changes or benefits of doing this at a smaller geographical area level?
I'm doing a proposal for a research to identify the target and mode of action of one drug against P. falciparum using OMICS and one of the strategies is to create a resistant strain from a sensitive one against that drug and check where the mutations occurred on the genes using omics and would like some advice about recommended techniques and protocols.
I'm working with RNA extraction from Plasmodium falciparum samples. I want to send the culture sample for my friend. Is it best to store the sample at TRIzol or RNA later and also for transportation?
Please help me with this. Thanks in advance.
I am isolating Plasmodium falciparum merozoite from 3D7 culture, I am doing sorbitol synchronisation and enrichment through MACS column, followed by maturation of parasite in mature schizont, Than I am passing mature schizont through 1.2 um syringe filter. Every steps are going fine, I am able to get merozoite at the end but it is in ~1 ml volume which is higher enough I want to concentrate in 50-100 ul volume. I tried multiple time to do this by spinning at different speed (up to 10000 rpm) but not successful and I lost larger proportion of merozoite by this process. Can any one share their protocols, even if it is different method. Or if there is optimised protocol that can be used to concentrate merozoite.
Up to my knowledge, P. falciparum dhfr mutant alleles are selected in a step-wise fashion, starting with a mutation at codon 108 from serine to asparagine (S108N) whereas that at codon 51 is a successive one. However, I do not know whether 51I could be existent alone without 108N. I would appreciate experts in the field for giving their input about this point.
I have a stock of RBC which is more than one month old but still my parasites are growing well in them as observed using giemsa staining. I'm planning for stable transfection. So can I use the same RBC (after washing step) or should I use fresh RBC.
Does change in batch of RBC change the growth of parasites.
Several approaches are available for the molecular detection of P. falciparum gametocytes. Can anyone kindly help me choose the best protocol? Any advice?!
I am doing the molecular characterization of Plasmodium species in the area of Kedougou (Senegal) and I would like to have some more information about the MSP-1 gene (chromosome location, allelic diversity).
Cells are P. falciparum freed from RBC's. This should be a quite fast method since I want to study transport processes.