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Accepted manuscript posted online 18 May 2009 , doi: 10.1128/IAI.01564-08

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Nasopharyngeal colonization represents the initial interaction between Haemophilus influenzae and its human host. Factors that influence bacterial carriage likely affect transmission and incidence of infection. Therefore, we investigated host factors involved in limiting H. influenzae colonization in BALB/c mice, as colonization can be established in this genetic background. Unlike what is observed in the C57BL/6 background, initial colonization of BALB/c mice was mainly limited by adaptive immune components. This effect on colonization did not require either CD4- or CD8-positive T cells. Instead, initial colonization by genetically diverse strains was limited by preexisting natural antibody with a lesser contribution of complement activity and the presence of neutrophils. Natural serum immunoglobulin from mice was able to bind to the bacterial surface and exhibited complement-dependent bactericidal activity against these genetically diverse H. influenzae strains. Moreover, natural immunoglobulin G (IgG) recognizing these strains was detected at the nasopharyngeal mucosal surface. This antibody-mediated effect required exposure to the normal mouse microbial flora, since mice raised under germfree (GF) conditions showed increased levels of H. influenzae colonization that were not limited by adaptive immunity. In addition, serum IgG from GF mice exhibited less surface binding to H. influenzae , suggesting that natural antibody, induced through prior exposure to the microbial flora, mediated the observed reduction in initial colonization. The broad effect of natural IgG against genetically diverse isolates suggests the presence of conserved species-wide protective targets of antibody.

Haemophilus influenzae is a gram-negative pathogen that commonly colonizes the mucosal surface of the human nasopharynx. This species is capable of causing a wide spectrum of diseases ranging from acute infection of the respiratory tract to sepsis and meningitis. Invasive infections were most commonly associated with the encapsulated type b strain ( H. influenzae type b [Hib]); however, the introduction of Hib conjugate polysaccharide vaccines has dramatically reduced the incidence of these infections. Conversely, nontypeable strains (nontypeable H. influenzae [NTHI]) do not express a polysaccharide capsule and are frequently associated with mucosal infections including otitis media, chronic bronchitis, and community-acquired pneumonia ( 1 , 11 , 26 ). The development of an effective vaccine to prevent NTHI infection has been hampered by the marked intra- and interstrain heterogeneity of surface antigens in this genetically diverse species.

We Should All Be Feminists by Chimamanda Ngozi Adichie (TED Talk: The danger of a single story ) At one point, I started calling myself a humanist instead of a feminist. I convinced myself by saying I cared about all humans, so why bother with those negative associations with the word “feminist”? I read this short book while I was on a plane, and who would have thought that after all these years, a book would make me call myself a feminist again with pride, genuineness and fearlessness? Adichie says she started calling herself “a happy African feminist who does not hate men and who likes to wear lip gloss and high heels for herself and not for men” to defend against the stereotypes associated with the f-word. Although I may not be African, do not wear lipstick often, and quickly get tired from high heels, I felt she was speaking about me. I felt she was in my (flat) shoes! To women and men, mothers and fathers, brothers and sisters, daughters and sons, I encourage you all to embrace being a feminist. — Lana Mazareh (TED Talk: 3 thoughtful ways to conserve water )

Sorry, Not Sorry by Haji Mohamed Dawjee This memoir is an intimate and funny unpacking of a brown woman’s experience of life in modern-day South Africa. Everything from sneakers to sexuality is covered in the most hilarious, heartwarming, thought-provoking and sometimes heartbreaking way. It’s bound to make people uncomfortable in all the best ways. — Tiffany Mugo (TED Talk with Siphumeze Khundayi: How to have a healthier, positive relationship to sex)

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by Clarissa Pinkola Estes “Stories are medicine,” writes Estes in her seminal work. As a poet and a playwright, I believe this to be true. The Wild Woman archetype is not the aspirational end product of constant activity and striving; she is our birthright — untamed, instinctive, innate. Through folktales and cultural narratives, the author illuminates the historic and present-day significance of the Wild Woman and reminds us that she is essential to the psychological and spiritual well-being of individual women and the world as a whole. To be clear, this is not a beach read. This is a sit-at-your-desk-highlighter-in-hand-read-a-few-pages-and-reflect kind of book. It’s certain to inspire. — Felice Belle (TED Talk with Jennifer Murphy: How we became sisters )

If I Stay Right Here: A Novel by Chwayita Ngamlana This book is a dark and extremely personal account of one woman’s experience of intimate partner violence with her girlfriend. The author uses an experimental literary style that makes reading it a bit of a trip sometimes. Intimate partner violence is not a topic that’s often spoken about, but this semi-biographical book breaks the silence. — Tiffany Mugo (TED Talk with Siphumeze Khundayi: How to have a healthier, positive relationship to sex)

Figure 1. ERRBS improves genomic coverage and alignment accuracy.

(A) Average CpG number coverage for ERRBS (red) and RRBS (green) methods (n = 3, samples NBM_#2, AML and MLLr_#1). (B) Average percent coverage of different genomic regions by ERRBS (red) and RRBS (green) (n = 3, samples NBM_#2, AML and MLLr_#1) (C) Average percentage of uniquely aligned reads using a whole genome reference strategy (black) or an MspI in silico digested genome reference (gray) (n = 4, samples NBM_#2, AML_Rep#2, MLLr_#1 and MLLr_#2) (D) Example of a misalignment due to the use of a reduced representation bisulfite converted reference genome. The read aligns to a unique genomic location using the MspI alignment algorithm (forward strand, chr1: 876391–876441), however the same fragment does not align uniquely when using a whole genome alignment algorithm, rather it also aligns to the reverse strand of chr 2: 130,704,784–130,704,833.

While the original RRBS alignment strategy used an MspI fragmented genome as a reference, whole-genome alignment strategies can also be applied to these data [18] . In a direct comparison of both strategies, we observed that a whole-genome alignment approach using the Bowtie aligner via the Bismark software Isabel Marant Elaya oversized jumper 100% Guaranteed For Sale Cheap Sale Fast Delivery H62cjdmyw0
more than doubled the number of aligned reads which resulted in an increased recovery of the number of CpGs (mean increase 200,154+/−135,012) ( Figure 1C ). Eliminating the use of an MspI site as the absolute alignment requirement at the beginning of reads, as well as the use of a longer (32 bp) seed length, further improved accuracy by excluding those reads that had the potential for more than one unique match or mismatch ( Figure 1D ). Theoretically, no reads should map to regions of the genome that are not flanked by an MspI restriction sites, yet we found that on average 29% of the aligned reads mapped to non-MspI fragments. These fragments, which would be discarded when using in silico digested genomes for alignment, were likely produced due to either restriction enzyme non-specific activity, the presence of partially degraded DNA at the onset of the protocol, or variations in the patient genome compared to the reference genome. Collectively these approaches enhanced not only genomic coverage, but also alignment efficiency and accuracy.

We previously reported that IDH-mut and MLLr AMLs distribute to different DNA methylation clusters and have distinct promoter DNA methylation signatures compared to normal CD34+ bone marrow controls (NBM) [10] , [11] . We performed ERRBS in two IDH-mut AML samples, two MLLr cases harboring t(9;11)(q22,q23) translocations, and two NBM samples. ERRBS covered an average of 2,082,426 CpGs per sample. In addition to the expected high correlation between the NBMs (r = 0.96) there was a remarkable degree of correlation between the two IDH patients (r = 0.93) and the two MLLr patients (r = 0.92) ( Figure S2 ), which far exceeded the correlation between MLLr and IDH-mut patients (r = 0.85–0.88), suggesting a strong link between genetic background and its effects on cytosine methylation. Unsupervised analyses using hierarchical clustering (1-Pearson correlation distance + Ward clustering method) and principal component analysis revealed that, even with this greatly enhanced representation of the genome, ERRBS methylation profiles from IDH-mut and MLLr naturally segregate from each other just as strongly as from NBM ( Figure 2A and Figure S3A ). In order to determine whether this natural segregation was driven solely by promoter differences in methylation, as captured in our previous studies, or whether biologically relevant differences were conserved in all genomic regions, we repeated the clustering analysis using only CpG sites within defined genomic regions. We found that using either (1) all non-promoter CpGs, (2) non-promoter intron CpGs, or (3) CpG sites at CpG islands and shores regardless of genomic location, the clustering results still retained the natural segregation into the biological groups ( Figure 2B and Figure S3B S3E ). Notably, when the clustering was performed on CpG island-associated CpG sites ( Figure S3D ), IDH-mut AMLs segregated further apart from NBMs and MLLr AMLs, indicating that these sites are likely to be more heavily involved in the aberrant DNA methylation profiles of these AMLs. These findings demonstrate the existence of robust AML subtype-specific DNA methylation patterns, which extend beyond promoters to include other genomic regions.

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